Compositions and methods for modulating growth hormone receptor expression

ABSTRACT

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with excess growth hormone using antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR).

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0253USC1SEQ_ST25.txt created Apr. 30, 2018, which is 1.29 MB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with excess growth hormone using antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR).

BACKGROUND

Growth hormone is produced in the pituitary and secreted into the bloodstream where it binds to growth hormone receptor (GHR) on many cell types, causing production of insulin-like growth factor-1 (IGF-1). IGF-1 is produced mainly in the liver, but also in adipose tissue and the kidney, and secreted into the bloodstream. Several disorders, such as acromegaly and gigantism, are associated with elevated growth hormone levels and/or elevated IGF-I levels in plasma and/or tissues.

Excessive production of growth hormone can lead to diseases such as acromegaly or gigantism. Acromegaly and gigantism are associated with excess growth hormone, often caused by a pituitary tumor, and affects 40-50 per million people worldwide with about 15,000 patients in each of the US and Europe and an annual incidence of about 4-5 per million people. Acromegaly and gigantism are initially characterized by abnormal growth of the hands and feet and bony changes in the facial features. Many of the growth related outcomes are mediated by elevated levels of serum IGF-1.

SUMMARY

Embodiments provided herein relate to methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with excess growth hormone. Several embodiments provided herein are drawn to antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR). Several embodiments are directed to treatment, prevention, or amelioration of acromegaly with antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR).

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^(st) edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

“2′-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine in a 2′-F nucleoside is in the ribo position (replacing the OH of a natural ribose).

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxy-ethyl modification at the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.

“2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.

“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.

“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within ±10% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of GHR”, it is implied that GHR levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

“Alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

“Bicyclic nucleic acid” or “ BNA” or “BNA nucleosides” means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“Carbohydrate” means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

“Carbohydrate cluster” means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

“Carbohydrate derivative” means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.

“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.

“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

“Cleavable bond” means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.

“Cleavable moiety” means a bond or group that is capable of being split under physiological conditions. In certain embodiments, a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as a lysosome. In certain embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.

“Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and/or clearance properties.

“Conjugate linker” or “linker” in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link (1) an oligonucleotide to another portion of the conjugate group or (2) two or more portions of the conjugate group.

Conjugate groups are shown herein as radicals, providing a bond for forming covalent attachment to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3′-oxygen atom of the 3′-hydroxyl group of the 3′ terminal nucleoside of the oligomeric compound. In certain embodiments the point of attachment on the oligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group of the 5′ terminal nucleoside of the oligomeric compound. In certain embodiments, the bond for forming attachment to the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster portion comprises: a targeting moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups and is designated “GalNAc₃”. In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated “GalNAc₄”. Specific carbohydrate cluster portions (having specific tether, branching and conjugate linker groups) are described herein and designated by Roman numeral followed by subscript “a”. Accordingly “GalNAc3-1_(a)” refers to a specific carbohydrate cluster portion of a conjugate group having 3 GalNAc groups and specifically identified tether, branching and linking groups. Such carbohydrate cluster fragment is attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.

“Conjugate compound” means any atoms, group of atoms, or group of linked atoms suitable for use as a conjugate group. In certain embodiments, conjugate compounds may possess or impart one or more properties, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′bridge.

“Deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

“Designing” or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

“Differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be liquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.

“Double-stranded” refers to two separate oligomeric compounds that are hybridized to one another. Such double stranded compounds may have one or more or non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.

“Downstream” refers to the relative direction towards the 3′ end or C-terminal end of a nucleic acid.

“Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Effective amount” in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of pharmaceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.

“Expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”

“Growth Hormone Receptor (GHR)” means any nucleic acid or protein of GHR. “GHR nucleic acid” means any nucleic acid encoding GHR. For example, in certain embodiments, a GHR nucleic acid includes a DNA sequence encoding GHR, an RNA sequence transcribed from DNA encoding GHR (including genomic DNA comprising introns and exons), including a non-protein encoding (i.e. non-coding) RNA sequence, and an mRNA sequence encoding GHR. “GHR mRNA” means an mRNA encoding a GHR protein.

“GHR specific inhibitor” refers to any agent capable of specifically inhibiting GHR RNA and/or GHR protein expression or activity at the molecular level. For example, GHR specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of GHR RNA and/or GHR protein.

“Halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

“Heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

“Identifying an animal having, or at risk for having, a disease, disorder and/or condition” means identifying an animal having been diagnosed with the disease, disorder and/or condition or identifying an animal predisposed to develop the disease, disorder and/or condition. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Internucleoside neutral linking group” means a neutral linking group that directly links two nucleosides.

“Internucleoside phosphorus linking group” means a phosphorus linking group that directly links two nucleosides.

“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.

“Linkage motif” means a pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Locked nucleic acid nucleoside” or “LNA” “Locked nucleic acid” or “ LNA” or “LNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4′ and 2′position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, as depicted below.

As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—, —C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—, —S(═O)_(x)— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R₁ and R₂ is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_(n)—, —[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—. Furthermore, other bridging groups encompassed with the definition of LNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′,4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′- bridges, wherein each R₁ and R₂ is, independently, H, a protecting group or C₁-C₁₂ alkyl.

Also included within the definition of LNA according to the invention are LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′ carbon atom of the sugar ring, thereby forming a methyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (—CH₂—) group connecting the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicylic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy (4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is also encompassed within the definition of LNA, as used herein.

“Metabolic disorder” means a disease or condition principally characterized by dysregulation of metabolism—the complex set of chemical reactions associated with breakdown of food to produce energy.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

“Modified carbohydrate” means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety. “Modified sugar moiety” means a substituted sugar moiety or a sugar surrogate.

“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating GHR mRNA can mean to increase or decrease the level of GHR mRNA and/or GHR protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, a GHR antisense compound can be a modulator that decreases the amount of GHR mRNA and/or GHR protein in a cell, tissue, organ or organism.

“MOE” means —OCH₂CH₂OCH₃.

“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Mono or polycyclic ring system” is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two =0 groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.

“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH). “Naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Neutral linking group” means a linking group that is not charged. Neutral linking groups include without limitation phosphotriesters, methylphosphonates, MMI (—CH₂—N(CH₃)—O—), amide-3 (—CH₂—C(═O)—N(H)—), amide-4 (—CH₂—N(H)—C(═O)—), formacetal (—O—CH₂—O—), and thioformacetal (—S—CH₂—O—). Further neutral linking groups include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)). Further neutral linking groups include nonionic linkages comprising mixed N, O, S and CH₂ component parts.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Non-internucleoside neutral linking group” means a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral linking group links two groups, neither of which is a nucleoside.

“Non-internucleoside phosphorus linking group” means a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

“Nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleoside motif” means a pattern of nucleoside modifications in an oligonucleotide or a region thereof. The linkages of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

“Oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. Oligomeric compounds also include naturally occurring nucleic acids. In certain embodiments, an oligomeric compound comprises a backbone of one or more linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. In certain embodiments, oligomeric compounds may also include monomeric subunits that are not linked to a heterocyclic base moiety, thereby providing abasic sites. In certain embodiments, the linkages joining the monomeric subunits, the sugar moieties or surrogates and the heterocyclic base moieties can be independently modified. In certain embodiments, the linkage-sugar unit, which may or may not include a heterocyclic base, may be substituted with a mimetic such as the monomers in peptide nucleic acids.

“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

“Peptide” means a molecular formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, peptide refers to polypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, a conjugated antisense oligonucleotide targeted to GHR is a pharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Phosphorus linking group” means a linking group comprising a phosphorus atom. Phosphorus linking groups include without limitation groups having the formula:

wherein:

R_(a) and R_(d) are each, independently, O, S, CH₂, NH, or NJ₁ wherein J₁ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, amino or substituted amino; and

J₁ is R_(b) is O or S.

Phosphorus linking groups include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound

“Prevent” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.

“Prodrug” means an inactive or less active form of a compound which, when administered to a subject, is metabolized to form the active, or more active, compound (e.g., drug).

“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

“Protecting group” means any compound or protecting group known to those having skill in the art. Non-limiting examples of protecting groups may be found in “Protective Groups in Organic Chemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“RISC based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to the RNA Induced Silencing Complex (RISC).

“RNase H based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.

“Salts” mean a physiologically and pharmaceutically acceptable salt of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Separate regions” means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.

“Sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

“Side effects” means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Single-stranded” means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Slows progression” means decrease in the development of the said disease.

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.

“Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substituent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present disclosure have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms that differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido (—C(O)N—(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido (—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl (—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl (—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol (—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) and sulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

“Substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.

“Sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

“Sugar motif” means a pattern of sugar modifications in an oligonucleotide or a region thereof.

“Sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds. “Target nucleic acid” means a nucleic acid molecule to which an antisense compound is intended to hybridize to result in a desired antisense activity. Antisense oligonucleotides have sufficient complementarity to their target nucleic acids to allow hybridization under physiological conditions.

“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

“Terminal internucleoside linkage” means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“The same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleosides have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

“Treat” refers to administering a pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal. In certain embodiments, one or more pharmaceutical compositions can be administered to the animal.

“Type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.

“Unmodified” nucleobases or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Upstream” refers to the relative direction towards the 5′ end or N-terminal end of a nucleic acid.

“Wing segment” means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Certain Embodiments

Certain embodiments provide methods, compounds and compositions for inhibiting growth hormone receptor (GHR) expression.

Certain embodiments provide antisense compounds targeted to a GHR nucleic acid. In certain embodiments, the GHR nucleic acid has the sequence set forth in GENBANK Accession No. NM_000163.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No X06562.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. DR_(006395.1) (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DB052048.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. AF230800.1 (incorporated herein as SEQ ID NO: 6), the complement of GENBANK Accession No. AA398260.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No. BC136496.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No. NM_001242399.2 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No. NM_001242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK Accession No. NM_001242401.3 (incorporated herein as SEQ ID NO: 11), GENBANK Accession No. NM_001242402.2 (incorporated herein as SEQ ID NO: 12), GENBANK Accession No. NM_001242403.2 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No. NM_001242404.2 (incorporated herein as SEQ ID NO: 14), GENBANK Accession No. NM_001242405.2 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No. NM_001242406.2 (incorporated herein as SEQ ID NO: 16), GENBANK Accession No. NM_001242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM_001242461.1 (incorporated herein as SEQ ID NO: 18), or GENBANK Accession No. NM_001242462.1 (incorporated herein as SEQ ID NO: 19).

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 9 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 10 contiguous nucleobases of the nucleobase sequences of any of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 11 contiguous nucleobases of the nucleobase sequences of any of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 12 contiguous nucleobases of the nucleobase sequences of any of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequences of any of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of the nucleobase sequences of any one of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleotides 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660 of SEQ ID NO: 1, wherein said modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases 100% complementary to an equal length portion of nucleobases 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleotides 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295 of SEQ ID NO: 2, wherein said modified oligonucleotide is at least 90% complementary to SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases 100% complementary to an equal length portion of nucleobases 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295 of SEQ ID NO: 2, wherein the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 2. In certain aspects, the compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides complementary within nucleotides 155594-155613, 72107-72126, 153921-153940, 159252-159267, 213425-213440, 153004-153019, 155597-155612, 248233-248248 of SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 20-2295.

In certain embodiments, a compound comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a growth hormone receptor nucleic acid and is complementary within the following nucleotide regions of SEQ ID NO: 1: 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660.

In certain embodiments, a compound comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a growth hormone receptor nucleic acid and targets the following nucleotide regions of SEQ ID NO: 1: 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660.

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a region of a growth hormone receptor nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a GHR nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660.

In certain embodiments, a compound comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a growth hormone receptor nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 2: 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, a compound comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a growth hormone receptor nucleic acid targets the following nucleotide regions of SEQ ID NO: 2:: 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a region of a growth hormone receptor nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a GHR nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 2:: 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to target intron 1 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 3058-144965 (intron 1) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 2 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 145047-208139 (intron 2) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 3 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 208206-267991 (intron 3) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 4 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 268122-274018 (intron 4) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 5 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 274192-278925 (intron 5) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 6 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 279105-290308 (intron 6) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 7 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 290475-292530 (intron 7) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 8 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 292622-297153 (intron 8) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 9 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 297224-297554 (intron 9) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified sugar. In certain aspects, at least one modified sugar comprises a 2′-O-methoxyethyl group. In certain aspects, at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH₃)—O-2′ group, a 4′-CH₂—O-2′ group, or a 4′-(CH₂)₂—O-2′group.

In certain aspects, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;         wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment and wherein each nucleoside of         each wing segment comprises a modified sugar.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 918, 479, 703, 1800, 1904, 2122, 2127, or 2194.

In certain aspects, the modified oligonucleotide has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 918, 479 or 703, wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of five linked nucleosides; and

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.

In certain aspects, the modified oligonucleotide has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 1800, 1904, 2122, 2127, or 2194, wherein the modified oligonucleotide comprises of nucleosides that have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises a single-stranded modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 918, 479 or 703, wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of five linked nucleosides; and

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises a single-stranded modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 16 linked nucleosides and has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 1800, 1904, 2122, 2127, or 2194, wherein the modified oligonucleotide comprises of nucleosides that have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR and a conjugate group. For instance, in certain embodiments, a compound comprises ISIS 532401and a conjugate group.

In any of the foregoing embodiments, the compound or oligonucleotide can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding growth hormone receptor.

In any of the foregoing embodiments, the nucleic acid encoding growth hormone receptor can comprise the nucleotide sequence of any one of SEQ ID NOs: 1-19.

In any of the foregoing embodiments, the compound or oligonucleotide can be single-stranded.

In any of the foregoing embodiments, the compound or oligonucleotide can be double-stranded.

In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

In certain embodiments, at least one modified internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, or 7 phosphodiester internucleoside linkages.

In certain embodiments, each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester intemucleoside linkage and a phosphorothioate internucleoside linkage.

In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.

In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase.

In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises at least one modified sugar.

In certain embodiments, the modified sugar is a 2′ modified sugar, a BNA, or a THP.

In certain embodiments, the modified sugar is any of a 2′-O-methoxyethyl, 2′-O-methyl, a constrained ethyl, a LNA, or a 3′-fluoro-HNA.

In certain embodiments, the compound comprises at least one 2′-O-methoxyethyl nucleoside, 2′-O-methyl nucleoside, constrained ethyl nucleoside, LNA nucleoside, or 3′-fluoro-HNA nucleoside.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of 10 linked deoxynucleosides;

a 5′ wing segment consisting of 5 linked nucleosides; and

a 3′ wing segment consisting of 5 linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 19 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides.

Certain embodiments provide compounds consisting of a conjugate group and a modified oligonucleotide (SEQ ID NO: 703) according to the following formula: mCes mCes Aes mCes mCes Tds Tds Tds Gds Gds Gds Tds Gds Ads Ads Tes Aes Ges mCes Ae; wherein,

A=an adenine,

mC=a 5-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc on the 5′ end. For instance, in certain embodiments, a compound comprises ISIS 532401 conjugated to GalNAc on the 5′ end. In further embodiments, the compound has the following chemical structure comprising or consisting of ISIS 532401 (SEQ ID NO: 703) with 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

wherein X is a conjugate group comprising GalNAc.

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. In further embodiments, a compound having the following chemical structure comprises or consists of ISIS 719223 (SEQ ID NO: 703) with a 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage or a phosphodiester linkage. In further embodiments, a compound having the following chemical structure comprises or consists of ISIS 719224 (SEQ ID NO: 703) with a 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage or a phosphodiester linkage. In further embodiments, a compound having the following chemical structure comprises or consists of ISIS 766720 (SEQ ID NO: 703) with a 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc. In further such embodiments, the compound comprises the sequence of ISIS 532401 (SEQ ID NO: 703) conjugated to GalNAc, and is represented by the following chemical structure:

wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R² together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and R¹ and R² are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—; and for each pair of R³ and R⁴ on the same ring, independently for each ring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³ and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—; and R⁵ is selected from H and —CH₃; and Z is selected from S⁻ and O⁻.

In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs disclosed in WO 2004/078922 and a conjugate group described herein. The nucleobase sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference herein. For example, a compound comprises an oligonucleotide (SEQ ID NO: 2336) disclosed in WO 2004/078922 conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage and has the following chemical structure:

For example, a compound comprises an oligonucleotide (SEQ ID NO: 2336) disclosed in WO 2004/078922 conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide compound is a phosphorothioate linkage or a phosphodiester linkage, and has the following chemical structure:

Certain embodiments provide a composition comprising the compound of any of the aforementioned embodiments or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain aspects, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain aspects, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain aspects, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature or about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.

Certain embodiments provide a method of treating a disease associated with excess growth hormone in a human comprising administering to the human a therapeutically effective amount of the compound or composition of any of the aforementioned embodiments, thereby treating the disease associated with excess growth hormone. In certain aspects, the disease associated with excess growth hormone is acromegaly. In certain aspects, the treatment reduces IGF-1 levels.

Certain embodiments provide a method of preventing a disease associated with excess growth hormone in a human comprising administering to the human a therapeutically effective amount of a compound or composition of any of the aforementioned embodiments, thereby preventing the disease associated with excess growth hormone. In certain embodiments, the disease associated with excess growth hormone is acromegaly.

Certain embodiments provide a method of reducing growth hormone receptor (GHR) levels in a human comprising administering to the human a therapeutically effective amount of the compound or composition of any of the aforementioned embodiments, thereby reducing GHR levels in the human. In certain aspects, the human has a disease associated with excess growth hormone. In certain aspects, the disease associated with excess growth hormone is acromegaly.

In certain aspects, the foregoing methods comprise co-administering the compound or composition and a second agent. In certain aspects, the compound or composition and the second agent are administered concomitantly.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a GHR nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.

Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides.“Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound targeted to a GHR nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.

i. ssRNA Compounds

In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5′-terminal end. In certain such embodiments, the 5′-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5′-phosphate). In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. Certain modified 5′-terminal nucleosides may be found in the art, for example in WO/2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound has Formula IIc:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula IIc to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certain embodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certain embodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certain embodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ are each, independently, H or halogen. In certain embodiments, Q₁ and Q₂ is H and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protected thiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R_(c) are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁), O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) or O(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ are each, independently, H or C₁-C₆ alkyl. In certain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certain embodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G is O(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2′-F and 2′-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB)_(x)A_(y) wherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

-   AABBAA; -   ABBABB; -   AABAAB; -   ABBABAABB; -   ABABAA; -   AABABAB; -   ABABAA; -   ABBAABBABABAA; -   BABBAABBABABAA; or -   ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5′ terminal nucleoside, such as those of formula IIc or IIe.

In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif:

-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q-(AB)_(x)A_(y)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)_(x)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating PO/PS 8 PS ii. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.

The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is GHR. In certain embodiment, the degradation of the targeted GHR is facilitated by an activated RISC complex that is formed with compositions of the invention.

Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC.

The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of several embodiments provided herein can target GHR by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., “hairpin” or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75,100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g., has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g., has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.

In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5′ phosphoryl group of a nucleotide becomes linked to the 2′ hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2′ position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, published on Apr. 29, 2004 as WO 2004/035765, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode growth hormone receptor (GHR) targetable with the compounds provided herein include, without limitation, the following: GENBANK Accession No. NM_000163.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No X06562.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. DR_(006395.1) (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DB052048.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. AF230800.1 (incorporated herein as SEQ ID NO: 6), the complement of GENBANK Accession No. AA398260.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No. BC136496.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No. NM_001242399.2 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No. NM_001242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK Accession No. NM_001242401.3 (incorporated herein as SEQ ID NO: 11), GENBANK Accession No. NM_001242402.2 (incorporated herein as SEQ ID NO: 12), GENBANK Accession No. NM_001242403.2 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No. NM_001242404.2 (incorporated herein as SEQ ID NO: 14), GENBANK Accession No. NM_001242405.2 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No. NM_001242406.2 (incorporated herein as SEQ ID NO: 16), GENBANK Accession No. NM_001242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM_001242461.1 (incorporated herein as SEQ ID NO: 18), GENBANK Accession No. NM_001242462.1 (incorporated herein as SEQ ID NO: 19), or GENBANK Accession No NW_001120958.1 truncated from nucleotides 4410000 to 4720000 (incorporated herein as SEQ ID NO: 2332).

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a GHR nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a GHR nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a GHR nucleic acid).

Non-complementary nucleobases between an antisense compound and a GHR nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a GHR nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a GHR nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a GHR nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a GHR nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a GHR nucleic acid, or specified portion thereof.

The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a GHR nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

In certain embodiments, oligonucleotides comprise one or more methylphosponate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosponate linkages. In certain embodiments, one methylphosponate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂ are each independently H, C₁-C₁₂ alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′- O(CH₂)₂OCH₃ substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R_(n)), where each R₁, R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as constrained ethyl or cEt) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see published International Application WO/2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Zhou et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 61/026,995 and 61/097,787; Published PCT International applications WO 1999/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; WO 2009/006478; WO 2010/036698; WO 2011/017521; WO 2009/067647; WO 20009/100320. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)3-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy (4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F) methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA and (K) vinyl BNA as depicted below:

wherein Bx is the base moiety and R is independently H, a protecting group, C₁-C₁₂ alkyl or C₁-C₁₂ alkoxy.

In certain embodiments, bicyclic nucleosides are provided having Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—, —CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O— CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_(c), M_(c)J_(d), SJ_(c), N₃, OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl or substituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) of are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═)OJ_(j), C(═)NJ_(j)J_(k), C(═O)J_(j), O—C(═)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F, O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:

In certain embodiments, sugar surrogates are selected having Formula VII:

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T_(a) and T_(b) is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T_(a) and T_(b) is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁ and R₂ is fluoro. In certain embodiments, R₁ is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′-or 3′-terminal group; and q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugar substituent group.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH₃ group at the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a GHR nucleic acid comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to a GHR nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Conjugated Antisense compounds

In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously. See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005). Such receptors are expressed on liver cells, particularly hepatocytes. Further, it has been shown that compounds comprising clusters of three N-acetylgalactosamine (GalNAc) ligands are capable of binding to the ASGP-R, resulting in uptake of the compound into the cell. See e.g., Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). Accordingly, conjugates comprising such GalNAc clusters have been used to facilitate uptake of certain compounds into liver cells, specifically hepatocytes. For example it has been shown that certain GalNAc-containing conjugates increase activity of duplex siRNA compounds in liver cells in vivo. In such instances, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex. Since the sense strand is discarded before the antisense strand ultimately hybridizes with the target nucleic acid, there is little concern that the conjugate will interfere with activity. Typically, the conjugate is attached to the 3′ end of the sense strand of the siRNA. See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups described herein are more active and/or easier to synthesize than conjugate groups previously described.

In certain embodiments of the present invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to RNase H based antisense compounds and antisense compounds that alter splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain attached to the antisense compound long enough to provide benefit (improved uptake into cells) but then should either be cleaved, or otherwise not interfere with the subsequent steps necessary for activity, such as hybridization to a target nucleic acid and interaction with RNase H or enzymes associated with splicing or splice modulation. This balance of properties is more important in the setting of single-stranded antisense compounds than in siRNA compounds, where the conjugate may simply be attached to the sense strand. Disclosed herein are conjugated single-stranded antisense compounds having improved potency in liver cells in vivo compared with the same antisense compound lacking the conjugate. Given the required balance of properties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavable moiety. As noted, without wishing to be bound by mechanism, it is logical that the conjugate should remain on the compound long enough to provide enhancement in uptake, but after that, it is desirable for some portion or, ideally, all of the conjugate to be cleaved, releasing the parent compound (e.g., antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments take advantage of endogenous nucleases in the cell by attaching the rest of the conjugate (the cluster) to the antisense oligonucleotide through a nucleoside via one or more cleavable bonds, such as those of a phosphodiester linkage. In certain embodiments, the cluster is bound to the cleavable nucleoside through a phosphodiester linkage. In certain embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three cleavable nucleosides. In such embodiments, such cleavable nucleosides are linked to one another, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage). Certain conjugates herein do not comprise a cleavable nucleoside and instead comprise a cleavable bond. It is shown that that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is vulnerable to cleavage in the cell (a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are ultimately metabolized to a more active form. For example, conjugated antisense compounds are cleaved to remove all or part of the conjugate resulting in the active (or more active) form of the antisense compound lacking all or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of an oligonucleotide. Certain such 5′-conjugates are cleaved more efficiently than counterparts having a similar conjugate group attached at the 3′ end. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, oligonucleotides comprising a conjugate at the 5′ end have greater efficacy than oligonucleotides comprising a conjugate at the 3′ end (see, for example, Examples 56, 81, 83, and 84). Further, 5′-attachment allows simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3′ to 5′ direction. To make a 3′-conjugated oligonucleotide, typically one attaches a pre-conjugated 3′ nucleoside to the solid support and then builds the oligonucleotide as usual. However, attaching that conjugated nucleoside to the solid support adds complication to the synthesis. Further, using that approach, the conjugate is then present throughout the synthesis of the oligonucleotide and can become degraded during subsequent steps or may limit the sorts of reactions and reagents that can be used. Using the structures and techniques described herein for 5′-conjugated oligonucleotides, one can synthesize the oligonucleotide using standard automated techniques and introduce the conjugate with the final (5′-most) nucleoside or after the oligonucleotide has been cleaved from the solid support.

In view of the art and the present disclosure, one of ordinary skill can easily make any of the conjugates and conjugated oligonucleotides herein. Moreover, synthesis of certain such conjugates and conjugated oligonucleotides disclosed herein is easier and/or requires few steps, and is therefore less expensive than that of conjugates previously disclosed, providing advantages in manufacturing. For example, the synthesis of certain conjugate groups consists of fewer synthetic steps, resulting in increased yield, relative to conjugate groups previously described. Conjugate groups such as GalNAc3-10 in Example 46 and GalNAc3-7 in Example 48 are much simpler than previously described conjugates such as those described in U.S. Pat. No. 8,106,022 or U.S. Pat. No. 7,262,177 that require assembly of more chemical intermediates. Accordingly, these and other conjugates described herein have advantages over previously described compounds for use with any oligonucleotide, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or two GalNAc ligands. As shown, such conjugates groups improve activity of antisense compounds. Such compounds are much easier to prepare than conjugates comprising three GalNAc ligands. Conjugate groups comprising one or two GalNAc ligands may be attached to any antisense compounds, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially alter certain measures of tolerability. For example, it is shown herein that conjugated antisense compounds are not more immunogenic than unconjugated parent compounds. Since potency is improved, embodiments in which tolerability remains the same (or indeed even if tolerability worsens only slightly compared to the gains in potency) have improved properties for therapy.

In certain embodiments, conjugation allows one to alter antisense compounds in ways that have less attractive consequences in the absence of conjugation. For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with phosphodiester linkages results in improvement in some measures of tolerability. For example, in certain instances, such antisense compounds having one or more phosphodiester are less immunogenic than the same compound in which each linkage is a phosphorothioate. However, in certain instances, as shown in Example 26, that same replacement of one or more phosphorothioate linkages with phosphodiester linkages also results in reduced cellular uptake and/or loss in potency. In certain embodiments, conjugated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the conjugated full-phosphorothioate counterpart. In fact, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides comprising a conjugate and at least one phosphodiester internucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since conjugation results in substantial increases in uptake/potency a small loss in that substantial gain may be acceptable to achieve improved tolerability. Accordingly, in certain embodiments, conjugated antisense compounds comprise at least one phosphodiester linkage.

In certain embodiments, conjugation of antisense compounds herein results in increased delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver tissue. However, in certain embodiments, that increased delivery alone does not explain the entire increase in activity. In certain such embodiments, more compound enters hepatocytes. In certain embodiments, even that increased hepatocyte uptake does not explain the entire increase in activity. In such embodiments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in hepatocytes versus non-parenchymal cells. This enrichment is beneficial for oligonucleotides that target genes that are expressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result in reduced kidney exposure. For example, as shown in Example 20, the concentrations of antisense oligonucleotides comprising certain embodiments of GalNAc-containing conjugates are lower in the kidney than that of antisense oligonucleotides lacking a GalNAc-containing conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the formula:

A-B-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group “D” branches as many times as is necessary to accommodate the number of (E-F) groups as indicated by “q”. Thus, where q=1, the formula is:

A-B-C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

In certain embodiments, conjugated antisense compounds are provided having the structure:

The present disclosure provides the following non-limiting numbered embodiments:

-   Embodiment 1. The conjugated antisense compound of any of     embodiments 1179 to 1182, wherein the tether has a structure     selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

-   Embodiment 2. The conjugated antisense compound of any of     embodiments 1179 to 1182, wherein the tether has the structure:

-   Embodiment 3. The conjugated antisense compound of any of     embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has a     structure selected from among:

-   Embodiment 4. The conjugated antisense compound of any of     embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has a     structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

-   Embodiment 5. The conjugated antisense compound of any of     embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has the     structure:

In embodiments having more than one of a particular variable (e.g., more than one “m” or “n”), unless otherwise indicated, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another.

i. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety attaches to the antisense oligonucleotide. In certain such embodiments, the cleavable moiety attaches directly to the cell-targeting moiety. In certain such embodiments, the cleavable moiety attaches to the conjugate linker. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is 2′-deoxy nucleoside that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2′-deoxy adenosine that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2′-deoxy adenosine that is attached to the 3′ position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to a 2′ position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached to the linker by either a phosphodiester or a phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after the complex has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved thereby releasing the active antisense oligonucleotide. While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more nucleases within the cell. In certain embodiments, the one or more nucleases cleave the phosphodiester linkage between the cleavable moiety and the linker. In certain embodiments, the cleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following:

ii. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. In certain such embodiments, the linker is covalently bound to the cleavable moiety. In certain such embodiments, the linker is covalently bound to the antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell-targeting moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support. In certain embodiments, the linker further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support and further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker further comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the linear group comprises groups selected from alkyl and ether groups. In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group includes at least one neutral linking group. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the cleavable moiety. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the antisense oligonucleotide. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety and a solid support. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety, a solid support and a protein binding moiety. In certain embodiments, the linear group includes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalently attached to a scaffold group. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide and ether groups. In certain embodiments, the scaffold includes at least one mono or polycyclic ring system. In certain embodiments, the scaffold includes at least two mono or polycyclic ring systems. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety and the linker. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a solid support. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a protein binding moiety. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker, a protein binding moiety and a solid support. In certain embodiments, the scaffold group includes one or more cleavable bond.

In certain embodiments, the linker includes a protein binding moiety. In certain embodiments, the protein binding moiety is a lipid such as for example including but not limited to cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein each L is, independently, a phosphorus linking group or a neutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

iii. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targeting moieties. Certain such cell-targeting moieties increase cellular uptake of antisense compounds. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, and one or more ligand. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, one or more ligand and one or more cleavable bond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group attaches the conjugate linker. In certain embodiments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group attaches the antisense oligonucleotide. In certain embodiments, the branching group is covalently attached to the linker and each of the tethered ligands. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the branching group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system. In certain embodiments, the branching group comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a branching group.

In certain embodiments, a branching group has a structure selected from among:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and     -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

-   -   wherein each A₁ is independently, O, S, CO═O or NH; and     -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

-   -   wherein A₁ is O, S, C═O or NH; and     -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

In certain embodiments, a branching group has a structure selected from among:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group through either an amide or an ether group. In certain embodiments, the tether is attached to the branching group through a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises from about 10 to about 18 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises about 13 atoms in chain length.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

-   -   wherein L is either a phosphorus linking group or a neutral         linking group;     -   Z₁ is C(═O)O—R₂;     -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;     -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and     -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and     -   each m₁ is, independently, from 0 to 20 wherein at least one m₁         is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group. In certain embodiments, a tether does not comprise any amide bonds. In certain embodiments, a tether comprises a phosphorus linking group and does not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands wherein each ligand is covalently attached to a tether. In certain embodiments, each ligand is selected to have an affinity for at least one type of receptor on a target cell. In certain embodiments, ligands are selected that have an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In certain embodiments, the targeting moiety comprises 3 N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, modified carbohydrate, multivalent carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, α-D-galactosamine, N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-β-D-glucopyranose, Methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNAc” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. In certain embodiments, “N-acetyl galactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNAc” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNAc” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these structures are intended to include the other form as well. For example, where the structure for an α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments, In certain preferred embodiments, the β-form 2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected from among:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected from among:

In certain embodiments one or more ligand has a structure selected from among:

In certain embodiments one or more ligand has a structure selected from among:

i. Certain Conjugates

In certain embodiments, conjugate groups comprise the structural features above. In certain such embodiments, conjugate groups have the following structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain such embodiments, conjugate groups have the following structure:

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of six to eleven consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of ten consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to eleven consecutively bonded atoms and wherein the tether comprises exactly one amide bond.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted or unsubstituted alkyl group, or a group comprising exactly one ether or exactly two ethers, an amide, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of eight consecutively bonded atoms, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein the tether comprises exactly one amide bond, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms and wherein the tether consists of an amide bond and a substituted or unsubstituted C₂-C₁₁ alkyl group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl group, or a group comprising an ether, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

wherein n is 4, 5, 6, 7, or 8.

In certain embodiments, conjugates do not comprise a pyrrolidine.

A Certain conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′, 3′, of 5′ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure:

A-B-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure:

A-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain such embodiments, the branching group comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′, 3′, of 5′ position of the nucleoside.

In certain embodiments, a conjugated antisense compound has the following structure:

A-B-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2′, 3′, of 5′ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure:

A-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure:

A-B-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure:

A-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc on the 5′ end. For instance, in certain embodiments, a compound comprises ISIS 532401 conjugated to GalNAc on the 5′ end. In further embodiments, the compound has the following chemical structure comprising or consisting of ISIS 532401 (SEQ ID NO: 703) with 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

wherein X is a conjugate group comprising GalNAc.

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. In further embodiments, the compound comprises the sequence of ISIS 532401 (SEQ ID NO: 703) conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. In such embodiments, the chemical structure is as follows:

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage or a phosphodiester linkage. In further embodiments, the compound comprises the sequence of ISIS 532401 (SEQ ID NO: 703) conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage or a phosphodiester linkage. In such embodiments, the chemical structure is as follows:

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc. In further such embodiments, the compound comprises the sequence of ISIS 532401 (SEQ ID NO: 703) conjugated to GalNAc, and is represented by the following chemical structure:

Wherein either R¹ is —OCH₂CH₂OCH₃ (MOE)and R² is H; or R¹ and R² together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and R¹ and R² are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And for each pair of R³ and R⁴ on the same ring, independently for each ring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³ and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And R⁵ is selected from H and —CH₃;

And Z is selected from S⁻ and O⁻.

Representative United States patents, United States patent application publications, and international patent application publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, U.S. Pat. No. 5,994,517, U.S. Pat. No. 6,300,319, U.S. Pat. No. 6,660,720, U.S. Pat. No. 6,906,182, U.S. Pat. No. 7,262,177, U.S. Pat. No. 7,491,805, U.S. Pat. No. 8,106,022, U.S. Pat. No. 7,723,509, U.S. 2006/0148740, U.S. 2011/0123520, WO 2013/033230 and WO 2012/037254, each of which is incorporated by reference herein in its entirety.

Representative publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, BIESSEN et al., “The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., “Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE et al., “New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes” Bioorganic & Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem. (1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNase H based oligonucleotide (such as a gapmer) or a splice modulating oligonucleotide (such as a fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain embodiments a conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998/013381; WO2011/038356; WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.

Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Certain Indications

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a disease associated with excess growth hormone in a subject by administering a GHR specific inhibitor, such as an antisense compound or oligonucleotide targeted to GHR. In certain aspects, the disease associated with excess growth hormone is acromegaly. In certain aspects, the disease associated with excess growth hormone is gigantism.

Certain embodiments provide a method of treating, preventing, or ameliorating acromegaly in a subject by administering a GHR specific inhibitor, such as an antisense compound or oligonucleotide targeted to GHR. Acromegaly is a disease associated with excess growth hormone (GH). In over 90 percent of acromegaly patients, the overproduction of growth hormones is caused by a benign tumor of the pituitary gland, called an adenoma, which produces excess growth hormone and compresses surrounding brain tissues. Expansion of the adenoma can cause headaches and visual impairment that often accompany acromegaly. In some instances, acromegaly is caused by tumors of the pancreas, lungs, or adrenal glands that lead to an excess of GH, either by producing GH or by producing Growth Hormone Releasing Hormone (GHRH), the hormone that stimulates the pituitary to make GH.

Acromegaly most commonly affects adults in middle age and can result in severe disfigurement, complicating conditions, and premature death. Because of its pathogenesis and slow progression, acromegaly often goes undiagnosed until changes in external features become noticeable, such as changes in the face. Acromegaly is often associated with gigantism.

Features of acromegaly include soft tissue swelling resulting in enlargement of the hands, feet, nose, lips and ears, and a general thickening of the skin; soft tissue swelling of internal organs, such as the heart and kidney; vocal cord swelling resulting in a low voice and slow speech; expansion of the skull; pronounced eyebrow protrusion, often with ocular distension; pronounced lower jaw protrusion and enlargement of the tongue; teeth gapping; and carpal tunnel syndrome. In certain embodiments, any one or combination of these features of acromegaly can be treated, prevented, or ameliorated by administering a compound or composition targeted to GHR provided herein.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Example 1 General Method for the Preparation of Phosphoramidites, Compounds 1, 1a and 2

Compounds 1, 1a and 2 were prepared as per the procedures well known in the art as described in the specification herein (see Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT International Applications (WO 2011/115818, WO 2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO 2007/090071), and U.S. Pat. No. 7,569,686).

-   -   Bx is a heterocyclic base;

Example 2 Preparation of Compound 7

Compounds 3 (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem., 1991, 34, 2692).

Example 3 Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4 Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 5 Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6 Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5.

Example 7 Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8 Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9 General Preparation of Conjugated ASOs Comprising GalNAc₃-1 at the 3′ Terminus, Compound 29

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-1 (GalNAc₃-1_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-1_(a) has the formula:

The solid support bound protected GalNAc₃-1, Compound 25, was prepared as per the procedures illustrated in Example 7. Oligomeric Compound 29 comprising GalNAc₃-1 at the 3′ terminus was prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 10 General Preparation Conjugated ASOs Comprising GalNAc₃-1 at the 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34 comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 11 Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustrated in Examples 2, 4, and 5. Compound 35 is prepared using similar procedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

Example 12 Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13 Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated in Examples 5 and 11. Compound 41 is prepared using similar procedures published in WO 2009082607.

Example 14 Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15 Preparation of Compound 47

Compound 46 is commercially available.

Example 16 Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared as per the procedures illustrated in Examples 4 and 15.

Example 17 Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18 Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19 General Method for the Preparation of Conjugated ASOs Comprising GalNAc₃-1 at the 3′ Position via Solid Phase Techniques (Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and ^(m)C residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on an GalNAc₃-1 loaded VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered 4 fold excess over the loading on the solid support and phosphoramidite condensation was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing dimethoxytrityl (DMT) group from 5′-hydroxyl group of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1:1 (v/v) mixture of triethylamine and acetonitrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesized using standard oligonucleotide synthesis procedures well known in the art.

Using these methods, three separate antisense compounds targeting ApoC III were prepared. As summarized in Table 17, below, each of the three antisense compounds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages; ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1 conjugated at its 3′end; and ISIS 647536 is the same as ISIS 647535 except that certain internucleoside linkages of that compound are phosphodiester linkages. As further summarized in Table 17, two separate antisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a 2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc₃-1 at its 3′-end.

TABLE 17 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed ID ASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds )T_(es)T_(es)T_(es)A_(es)T_(e) ApoC 7165.4 7164.4 2296 304801 III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′)- ApoC 9239.5 9237.8 2297 647535 GalNAc ₃-1 _(a) III ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′)- ApoC 9142.9 9140.8 2297 647536 GalNAc ₃-1 _(a) III ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) SRB-1 4647.0 4646.4 2298 440762 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′)-GalNAc ₃-1 _(a) SRB-1 6721.1 6719.4 2299 651900

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having the structure shown previously in Example 9. Note that GalNAc₃-1 comprises a cleavable adenosine which links the ASO to remainder of the conjugate, which is designated “GalNAc₃-1_(a).” This nomenclature is used in the above table to show the full nucleobase sequence, including the adenosine, which is part of the conjugate. Thus, in the above table, the sequences could also be listed as ending with “GalNAc₃-1” with the “A_(do)” omitted. This convention of using the subscript “a” to indicate the portion of a conjugate group lacking a cleavable nucleoside or cleavable moiety is used throughout these Examples. This portion of a conjugate group lacking the cleavable moiety is referred to herein as a “cluster” or “conjugate cluster” or “GalNAc₃ cluster.” In certain instances it is convenient to describe a conjugate group by separately providing its cluster and its cleavable moiety.

Example 20 Dose-Dependent Antisense Inhibition of Human ApoC III in huApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25 or 6.75 μmol/kg, or with PBS as a control. Each treatment group consisted of 4 animals. Forty-eight hours after the administration of the last dose, blood was drawn from each mouse and the mice were sacrificed and tissues were collected.

ApoC III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of ApoC III mRNA levels for each treatment group, normalized to PBS-treated control and are denoted as “% PBS”. The half maximal effective dosage (ED₅₀) of each ASO is also presented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relative to the PBS control. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Inter- Dose ED₅₀ nucleoside (μmol/ % (μmol/ 3′ linkage/ SEQ ASO kg) PBS kg) Conjugate Length ID No. PBS 0 100 — — — ISIS 0.08 95 0.77 None PS/20 2296 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074 GalNAc₃-1 PS/20 2297 647535 0.75 15 2.25 17 6.75 8

ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using procedures reported by Graham et al, Circulation Research, published online before print Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed without dilution using an Olympus Clinical Analyzer and a commercially available turbidometric ApoC III assay (Kamiya, Cat #KAI-006, Kamiya Biomedical, Seattle, Wash.). The assay protocol was performed as described by the vendor.

As shown in the Table 19 below, both antisense compounds reduced ApoC III protein relative to the PBS control. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Inter- Dose ED₅₀ nucleoside (μmol/ % (μmol/ 3′ Linkage/ SEQ ASO kg) PBS kg) Conjugate Length ID No. PBS 0 100 — — — ISIS 0.08 86 0.73 None PS/20 2296 304801 0.75 51 2.25 23 6.75 13 ISIS 0.08 72 0.19 GalNAc₃-1 PS/20 2297 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol. 37: 911-917, 1959) (Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959) (Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical analyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice and are denoted as “% PBS”. Results are presented in Table 20. As illustrated, both antisense compounds lowered triglyceride levels. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 20 Effect of ASO treatment on triglyceride levels in transgenic mice Inter- Dose ED₅₀ nucleoside (μmol/ % (μmol/ 3′ Linkage/ SEQ ASO kg) PBS kg) Conjugate Length ID No. PBS 0 100 — — — ISIS 0.08 87 0.63 None PS/20 2296 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc₃-1 PS/20 2297 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and of different fractions of cholesterol (HDL and LDL). Results are presented in Tables 21 and 22. As illustrated, both antisense compounds lowered total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoC III.

TABLE 21 Effect of ASO treatment on total cholesterol levels in transgenic mice Total Dose Cholesterol 3′ Internucleoside SEQ ASO (μmol/kg) (mg/dL) Conjugate Linkage/Length ID No. PBS 0    257 — — ISIS 0.08 226 None PS/20 2296 304801 0.75 164 2.25 110 6.75  82 ISIS 0.08 230 GalNAc₃-1 PS/20 2297 647535 0.75  82 2.25  86 6.75  99

TABLE 22 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice Inter- Dose nucleoside (μmol/ HDL LDL 3′ Linkage/ SEQ ASO kg) (mg/dL) (mg/dL) Conjugate Length ID No. PBS 0 17 28 — — ISIS 0.08 17 23 None PS/20 2296 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS 0.08 21 21 GalNAc₃-1 PS/20 2297 647535 0.75 44 2 2.25 50 2 6.75 58 2

Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were analyzed on MSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS 304801 and 647535 was measured and the results are provided in Table 23. As illustrated, liver concentrations of total full-length antisense compounds were similar for the two antisense compounds. Thus, even though the GalNAc₃-1-conjugated antisense compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated EC₅₀ (provided in Table 23) confirms that the observed increase in potency of the conjugated compound cannot be entirely attributed to increased accumulation. This result suggests that the conjugate improved potency by a mechanism other than liver accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.

The results also show that the concentration of GalNAc₃-1 conjugated antisense compound in the kidney is lower than that of antisense compound lacking the GalNAc conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired. These data suggest that GalNAc₃-1 conjugation reduces kidney accumulation.

TABLE 23 PK analysis of ASO treatment in transgenic mice Dose Liver Kidney Liver EC₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) (μg/g) (μg/g) (μg/g) Conjugate Linkage/Length ID No. ISIS 0.1 5.2 2.1 53 None PS/20 2296 304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.8 0.7 3.8 GalNAc₃-1 PS/20 2297 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8 237.2 179.3

Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass spectrometry analysis. The cleavage sites and structures of the observed metabolites are shown below. The relative % of full length ASO was calculated using standard procedures and the results are presented in Table 23a. The major metabolite of ISIS 647535 was full-length ASO lacking the entire conjugate (i.e. ISIS 304801), which results from cleavage at cleavage site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other cleavable bonds such as esters, peptides, disulfides, phosphoramidates or acyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can be cleaved by enzymes inside the cell, or which may cleave in the reductive environment of the cytosol, or which are labile to the acidic pH inside endosomes and lyzosomes, can also be useful.

TABLE 23a Observed full length metabolites of ISIS 647535 Cleavage Relative Metabolite ASO site % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3 GalNAc + 1 5-hydroxy-pentanoic acid tether] 5 ISIS 647535 minus D  9.9 [2 GalNAc + 2 5-hydroxy-pentanoic acid tether] 6 ISIS 647535 minus D  9.8 [3 GalNAc + 3 5-hydroxy-pentanoic acid tether]

Example 21 Antisense Inhibition of Human ApoC III in Human ApoC III Transgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III and described in Table 17, were further evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once at the dosage shown below with ISIS 304801, 647535 or 647536 (described above) or with PBS treated control. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA and protein levels in the liver; plasma triglycerides; and cholesterol, including HDL and LDL fractions were assessed, as described above (Example 20). Data from those analyses are presented in Tables 24-28, below. Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. The ALT and AST levels showed that the antisense compounds were well tolerated at all administered doses.

These results show improvement in potency for antisense compounds comprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1 conjugate and some phosphodiester linkages was as potent as ISIS 647535, which comprises the same conjugate, and all the internucleoside linkages within the ASO are phosphorothioate.

TABLE 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Inter- nucleoside Dose % ED₅₀ 3′ linkage/ SEQ ASO (mg/kg) PBS (mg/kg) Conjugate Length ID No. PBS 0 99 — — — ISIS 1 104 13.2 None PS/20 2296 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc₃-1 PS/20 2297 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc₃-1 PS/PO/20 2297 647536 1 60 3 31 10 21

TABLE 25 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III transgenic mice Inter- nucleoside Dose % ED₅₀ 3′ Linkage/ SEQ ASO (mg/kg) PBS (mg/kg) Conjugate Length ID No. PBS 0 99 — — — ISIS 1 104 23.2 None PS/20 2296 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1 GalNAc₃-1 PS/20 2297 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc₃-1 PS/PO/20 2297 647536 1 60 3 31 10 21

TABLE 26 Effect of ASO treatment on triglyceride levels in transgenic mice Inter- nucleoside Dose % ED₅₀ 3′ Linkage/ SEQ ASO (mg/kg) PBS (mg/kg) Conjugate Length ID No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/20 2296 304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc₃-1 PS/20 2297 647535 1 70 3 34 10 23 ISIS 0.3 95 1.9 GalNAc₃-1 PS/PO/20 2297 647536 1 66 3 31 10 23

TABLE 27 Effect of ASO treatment on total cholesterol levels in transgenic mice Dose 3′ Internucleoside SEQ ASO (mg/kg) % PBS Conjugate Linkage/Length ID No. PBS 0 96 — — ISIS 1 104 None PS/20 2296 304801 3 96 10 86 30 72 ISIS 0.3 93 GalNAc₃-1 PS/20 2297 647535 1 85 3 61 10 53 ISIS 0.3 115 GalNAc₃-1 PS/PO/20 2297 647536 1 79 3 51 10 54

TABLE 28 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice HDL LDL Dose % % 3′ Internucleoside SEQ ASO (mg/kg) PBS PBS Conjugate Linkage/Length ID No. PBS 0 131 90 — — ISIS 1 130 72 None PS/20 2296 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3 98 86 GalNAc₃-1 PS/20 2297 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc₃-1 PS/PO/20 2297 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency of an antisense compound. The results also show equal potency of a GalNAc₃-1 conjugated antisense compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six phosphodiester linkages) and a full phosphorothioate version of the same antisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisense compounds. For example, they resist nuclease digestion and they bind proteins resulting in accumulation of compound in the liver, rather than in the kidney/urine. These are desirable properties, particularly when treating an indication in the liver. However, phosphorothioate linkages have also been associated with an inflammatory response. Accordingly, reducing the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, but also lower concentration of the compound in liver, increase concentration in the kidney and urine, decrease stability in the presence of nucleases, and lower overall potency. The present results show that a GalNAc₃-1 conjugated antisense compound where certain phosphorothioate linkages have been replaced with phosphodiester linkages is as potent against a target in the liver as a counterpart having full phosphorothioate linkages. Such compounds are expected to be less proinflammatory (See Example 24 describing an experiment showing reduction of PS results in reduced inflammatory effect).

Example 22 Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1 In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17, were evaluated in a dose-dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNA levels. Further, the antisense compound comprising the GalNAc₃-1 conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 440762). These results demonstrate that the potency benefit of GalNAc₃-1 conjugates are observed using antisense oligonucleotides complementary to a different target and having different chemically modified nucleosides, in this instance modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar moiety).

TABLE 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice Internu- cleoside Dose Liver % ED₅₀ 3′ linkage/ SEQ ASO (mg/kg) PBS (mg/kg) Conjugate Length ID No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/14 2298 440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc₃-1 PS/14 2299 651900 0.2 63 0.7 20 2 6 7 5

Example 23 Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol

The hPBMC assay was performed using BD Vautainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat. #BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A 60 μl sample was pipette into a sample vial (Beckman Coulter) with 600 μl VersaLyse reagent (Beckman Coulter Cat #A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 μl/well of 96-well tissue culture plate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 μl/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).

Example 24 Evaluation of Proinflammatory Effects in hPBMC Assay for GalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluated for proinflammatory effect in hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for IL-6 release in the assay. The hPBMCs were isolated from fresh, volunteered donors and were treated with ASOs at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hr treatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ and E_(max) was calculated using standard procedures. Results are expressed as the average ratio of E_(max)/EC₅₀ from two donors and is denoted as “E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in the proinflammatory response and the higher ratio indicates a relative increase in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compound was the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO, ISIS 647535 was slightly less proinflammatory than its non-conjugated counterpart ISIS 304801. These results indicate that incorporation of some PO linkages reduces proinflammatory reaction and addition of a GalNAc₃-1 conjugate does not make a compound more proinflammatory and may reduce proinflammatory response. Accordingly, one would expect that an antisense compound comprising both mixed PS/PO linkages and a GalNAc₃-1 conjugate would produce lower proinflammatory responses relative to full PS linked antisense compound with or without a GalNAc₃-1 conjugate. These results show that GalNAc₃₋1 conjugated antisense compounds, particularly those having reduced PS content are less proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound, particularly one with reduced PS content, can be administered at a higher dose than a counterpart full PS antisense compound lacking a GalNAc₃-1 conjugate. Since half-life is not expected to be substantially different for these compounds, such higher administration would result in less frequent dosing. Indeed such administration could be even less frequent, because the GalNAc₃-1 conjugated compounds are more potent (See Examples 20-22) and re-dosing is necessary once the concentration of a compound has dropped below a desired level, where such desired level is based on potency.

TABLE 30 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISIS G_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds) TNFα 2300 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 2301 353512 G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 2296 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds )T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 2297 647535 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃-1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 2296 616468 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “A_(do′)-GalNAc₃-1_(a)” indicates a conjugate having the structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of the antisense oligonucleotide, as indicated.

TABLE 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMC assay Internu- cleoside EC₅₀ E_(max) E_(max)/ 3′ Linkage/ SEQ ASO (μM) (μM) EC₅₀ Conjugate Length ID No. ISIS 0.01 265.9 26,590 None PS/20 2301 353512 (high responder) ISIS 0.07 106.55 1,522 None PS/20 2296 304801 ISIS 0.12 138 1,150 GalNAc₃-1 PS/20 2297 647535 ISIS 0.32 71.52 224 None PS/PO/20 2296 616468

Example 25 Effect of GalNAc₃-1 Conjugated Modified ASO Targeting Human ApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells from transgenic mice at a density of 25,000 cells per well were treated with 0.03,0.08, 0.24, 0.74, 2.22, 6.67 and 20 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the hApoC III mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results are presented in Table 32. As illustrated, comparable potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.

TABLE 32 Modified ASO targeting human ApoC III in primary hepatocytes Internucleoside SEQ ASO IC₅₀ (μM) 3′ Conjugate linkage/Length ID No. ISIS 0.44 None PS/20 2296 304801 ISIS 0.31 GalNAc₃-1 PS/20 2297 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugation that are observed in vivo were not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates relative to oligonucleotides that lack the GalNAc conjugate (see Examples 60, 82, and 92).

Example 26 Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or with PBS treated control once per week for two weeks. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III protein levels in the liver as described above (Example 20). Data from those analyses are presented in Table 33, below.

These results show reduction in potency for antisense compounds with PO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33 Effect of ASO treatment on ApoC III protein levels in human ApoC III transgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) % PBS Conjugate linkage/Length No. PBS 0 99 — — ISIS 25 24 None Full PS 2296 304801 mg/kg/wk for 2 wks ISIS 25 40 None 14 PS/6 PO 2296 616468 mg/kg/wk for 2 wks

Example 27 Compound 56

Compound 56 is commercially available from Glen Research or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28 Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2. Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.

Compound 57 is meant to be representative and not intended to be limiting as other monoprotected substituted or unsubstituted alkyl diols including but not limited to those presented in the specification herein can be used to prepare phosphoramidites having a predetermined composition.

Example 29 Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. Org. Chem., 2013, 3, 566-577; and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to those reported in scientific and patent literature by Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., published PCT International Application, WO 2004063208.

Example 30 Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31 Preparation of Compound 63d

Compound 63d is prepared using procedures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32 Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 65 is prepared using procedures similar to those reported by Or et al., published PCT International Application, WO 2009003009. The protecting groups used for Compound 65 are meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 33 Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 68 is commercially available. The protecting group used for Compound 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 34 Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35 Preparation of Compound 79

Compound 76 was prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36 Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37 General Method for the Preparation of Conjugated Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus via Solid Support (Method I)

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2 (GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56 and 60 were prepared as per the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks including but not limited those presented in the specification herein can be used to prepare an oligomeric compound having a phosphodiester linked conjugate group at the 5′ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 38 Alternative Method for the Preparation of Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 cluster phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example 35. This alternative method allows a one-step installation of the phosphodiester linked GalNAc₃-2 conjugate to the oligomeric compound at the final step of the synthesis. The phosphoramidites illustrated are meant to be representative and not intended to be limiting, as other phosphoramidite building blocks including but not limited to those presented in the specification herein can be used to prepare oligomeric compounds having a phosphodiester conjugate at the 5′ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 39 General Method for the Preparation of Oligomeric Compound 83 h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1 Modified for 5′ End Attachment) via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4. Compounds 83a and 83b are commercially available. Oligomeric Compound 83e comprising a phosphodiester linked hexylamine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia provided the 5′-GalNAc₃-3 conjugated oligomeric compound (83 h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3 (GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40 General Method for the Preparation of Oligomeric Compound 89 Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′ Terminus via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4 (GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 is commercially available. Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are prepared as per the procedures illustrated in Examples 28 and 36. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a phosphodiester linked conjugate at the 3′ terminus with a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 41 General Method for the Preparation of ASOs Comprising a Phosphodiester Linked GalNAc₃-2 (See Example 37, Bx is Adenine) Conjugate at the 5′ Position via Solid Phase Techniques (Preparation of ISIS 661134)

Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and ^(m)C residues. Phosphoramidite compounds 56 and 60 were used to synthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered at a 4 fold excess over the initial loading of the solid support and phosphoramidite coupling was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing the dimethoxytrityl (DMT) groups from 5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during the coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 20% diethylamine in toluene (v/v) with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

TABLE 34 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ position targeting SRB-1 Observed SEQ ID ISIS No. Sequence (5′ to 3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) 6482.2 6481.6 2302 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of GalNAc₃-2_(a) is shown in Example 37.

Example 42 General Method for the Preparation of ASOs Comprising a GalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises a GalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

TABLE 34a ASO comprising a GalNAc₃-3 conjugate at the 5′ position via a hexylamino phosphodiester linkage targeting Malat-1 ISIS Calcd Observed No. Sequence (5′ to 3′) Conjugate Mass Mass SEQ ID No. 661166 5′-GalNAc ₃ -3 _(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) 5′-GalNAc ₃ -3 8992.16 8990.51 2303 ^(m)C_(ds)A_(ds)A_(ds)G_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “5′-GalNAc₃-3a” is shown in Example 39.

Example 43 Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (See Examples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 In Vivo

ISIS 661134 (see Example 41) comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9) were included in the study for comparison and are described previously in Table 17.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 661134 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

As illustrated in Table 35, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1 conjugate linked at the 3′ terminus (ISIS 651900) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was equipotent compared to ISIS 651900, which comprises the GalNAc₃-1 conjugate at the 3′ terminus.

TABLE 35 ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1 SRB-1 mRNA ISIS Dosage levels ED₅₀ SEQ ID No. (mg/kg) (% PBS) (mg/kg) Conjugate No. PBS 0 100 — — 440762 0.2 116 2.58 No conjugate 2298 0.7 91 2 69 7 22 20 5 651900 0.07 95 0.26 3′ GalNAc₃-1 2299 0.2 77 0.7 28 2 11 7 8 661134 0.07 107 0.25 5′ GalNAc₃-2 2302 0.2 86 0.7 28 2 10 7 6

Structures for 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previously in Examples 9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same manner as illustrated in Example 20. Liver sample was minced and extracted using standard protocols. The full length metabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) were identified and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a detectable level, were observed. Unlike its counterpart, additional metabolites similar to those reported previously in Table 23a were observed for the ASO having the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 651900). These results suggest that having the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate may improve the PK profile of ASOs without compromising their potency.

Example 44 Effect of PO/PS Linkages on Antisense Inhibition of ASOs Comprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ Terminus Targeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′ terminus each targeting SRB-1 were tested in a single administration study for their ability to inhibit SRB-1 in mice. The parent unconjugated compound, ISIS 353382 was included in the study for comparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten 2′-deoxyribonucleosides and each wing region comprises five 2′-MOE modified nucleosides. The ASOs were prepared using similar methods as illustrated previously in Example 19 and are described Table 36, below.

TABLE 36 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminus targeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no conjugate 2304 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO with 2305 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “GalNAc₃-1” is shown in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 655862 or with PBS treated control. Each treatment group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides comprising the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showed substantial improvement in potency comparing to the unconjugated antisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixed PS/PO linkages showed an improvement in potency relative to full PS (ISIS 655861).

TABLE 37 Effect of PO/PS linkages on antisense inhibition of ASOs comprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 SRB-1 mRNA ISIS Dosage levels ED₅₀ SEQ ID No. (mg/kg) (% PBS) (mg/kg) Chemistry No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS 2304 (parent) 10 52.40 without 30 24.95 conjugate 655861 0.5 81.22 2.2 Full PS with 2305 1.5 63.51 GalNAc₃-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3 Mixed PS/PO 2305 1.5 45.78 with 5 19.70 GalNAc₃-1 15 12.90 conjugate

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Organ weights were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 38) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS control. Further, the ASO with mixed PS/PO linkages (ISIS 655862) showed similar transaminase levels compared to full PS (ISIS 655861).

TABLE 38 Effect of PO/PS linkages on transaminase levels of ASOs comprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISIS Dosage ALT AST SEQ ID No. (mg/kg) (U/L) (U/L) Chemistry No. PBS 0 28.5 65 — 353382 3 50.25 89 Full PS 2304 (parent) 10 27.5 79.3 without 30 27.3 97 conjugate 655861 0.5 28 55.7 Full PS with 2305 1.5 30 78 GalNAc₃-1 5 29 63.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO 2305 1.5 21.7 58.5 with 5 29.3 69 GalNAc₃-1 15 22 61

Example 45 Preparation of PFP Ester, Compound 110a

Compound 4 (9.5g, 28.8 mmoles) was treated with compound 103a or 103b (38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves in dichloromethane (200 mL), and stirred for 16 hours at room temperature. At that time, the organic layer was filtered thru celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced under reduced pressure. The resultant oil was purified by silica gel chromatography (2%→10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was consistent with the structure.

Compounds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 105a and 105b in >90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90 under the same conditions as for compounds 901a-d, to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 106a and 106b were treated to the same conditions as for compounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as for compounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 109a and 109b in >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was consistent with the structure. Alternatively, Compound 110b can be prepared in a similar manner starting with Compound 109b.

Example 46 General Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5′-hexylamino modified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃ cluster dissolved in DMSO (50 μL) was added. If the PFP ester precipitated upon addition to the ASO solution DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h of mixing at room temperature. The resulting solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was then lyophilized to dryness and redissolved in concentrated aqueous ammonia and mixed at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to provide the GalNAc₃ conjugated oligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10 (GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 μmol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 μL) to fully dissolve the PFP ester. The reaction was complete after 16 h of mixing at room temperature. The solution was diluted with water to 12 mL total volume and spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous ammonia with mixing at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7 μmol).

TABLE 38a GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′) 5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o)A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) Hexylamine 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) GalNAc ₃ -10 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47 Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added, along with N,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction was allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%→10% methanol/dichloromethane) to give compounds 92a and 92b in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL of dichloromethane and 20 mL of trifluoroacetic acid at room temperature for 16 hours. The resultant solution was evaporated and then dissolved in methanol and treated with DOWEX-OH resin for 30 minutes. The resultant solution was filtered and reduced to an oil under reduced pressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7g, 9.6 mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 mmoles), and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%→20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated over Raney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalyst was removed by filtration thru celite, and the ethanol removed under reduced pressure to give compounds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structure.

Compound 23 (0.32g, 0.53 mmoles) was treated with HBTU (0.2g, 0.53 mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30 mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%→20% methanol/dichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent with the structure.

Compound 99 (0.17g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76 mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50 mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%→20% methanol/dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10% Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At that time, the catalyst was removed by filtration thru celite, and the organics removed under reduced pressure to give compounds 101a-d in 80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%→5% methanol/dichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48 Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate (22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite and washed the pad with 1:1 methanol/ethyl acetate. The filtrate and the washings were combined and concentrated to dryness to yield Compound 105a (quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction was stirred at room temperature for 6 h. Solvent was removed under reduced pressure to get an oil. The residue was dissolved in CH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100 mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20% MeOH in dichloromethane to yield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethyl acetate (4 mL/4 mL). Palladium on carbon (wet, 0.14 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added and the reaction was stirred at room temperature for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in CH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solution and brine and dried over anhydrous Na₂SO₄ and filtered. The organic layer was concentrated to dryness and the residue obtained was purified by silica gel column chromatography and eluted with 3 to 15% MeOH in dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl acetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHCO₃ solution. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na₂SO₄. The dichloromethane solution was concentrated to dryness and purified with silica gel column chromatography and eluted with 5 to 10% MeOH in dichloromethane to yield compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49 Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 min. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added and the reaction mixture was stirred for 18 h under an argon atmosphere. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf=0.47). The solvent was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150 mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL). Organic layer was dried with Na₂SO₄. Drying agent was removed by filtration and organic layer was concentrated by rotary evaporation. Crude mixture was purified by silica gel column chromatography and eluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g, 78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Mass m/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled solution of Compound 122 (7.75 g,13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The remaining solution was cooled an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon standing overnight. The organic layer was separated dried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. Product was used without any further purification. M.W.cal:574.36; M.W.fd:575.3 [M+H]⁺.

Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) and the resulting reaction mixture was cooled in an ice bath. To this N,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere. The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was completed after 1 h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1 M NaHSO₄ (3×100 mL), aqueous saturated NaHCO₃ (3×100 mL) and brine (2×100 mL). The organic phase separated dried (Na₂SO₄), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹H NMR were consistent with structure. Mass m/z 778.4 [M+H]⁺.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room temperature for 1 h. The reaction mixture was co-evaporated with toluene (30 mL) under reduced pressure to dryness. The residue obtained was co-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128 (1.67 g) as trifluoro acetate salt and used for next step without further purification. LCMS and ¹H NMR were consistent with structure. Mass m/z 478.2 [M+H]⁺.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅ under reduced pressure in a round bottom flask and then dissolved in anhydrous DMF (1 mL) and stirred for 5 min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture was stirred at room temperature under an argon atmosphere. The reaction was complete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO₄ (3×20 mL), aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organic phase was separated, dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography using 5-15% MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and ¹H NMR are consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20 mL scintillation vial. To this was added a small amount of 10% Pd/C (0.015 mg) and the reaction vessel was flushed with H₂ gas. The reaction mixture was stirred at room temperature under H₂ atmosphere for 18 h. The reaction mixture was filtered through a pad of Celite and the Celite pad was washed with methanol. The filtrate washings were pooled together and concentrated under reduced pressure to yield Compound 130 (0.08 g). LCMS and ¹H NMR were consistent with structure. The product was used without further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To this solution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 μL) drop wise with stirring. The reaction was completed after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHCl₃ (˜10 mL). The organic layer was partitioned against NaHSO₄ (1 M, 10 mL), aqueous saturated NaHCO₃ (10 mL) and brine (10 mL) three times each. The organic phase separated and dried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without further purification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50 Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

Synthesis of Compound 134: To a Merrifield flask was added aminomethyl VIMAD resin (2.5 g, 450 μmol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was swelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100 mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solution was allowed to stir for 5 min and was then added to the Merrifield flask with shaking. The suspension was allowed to shake for 3 h. The reaction mixture was drained and the resin was washed with acetonitrile, DMF and DCM. New resin loading was quantitated by measuring the absorbance of the DMT cation at 500 nm (extinction coefficient=76000) in DCM and determined to be 238 μmol/g. The resin was capped by suspending in an acetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterative Fmoc-based solid phase peptide synthesis methods. A small amount of solid support was withdrawn and suspended in aqueous ammonia (28-30 wt %) for 6 h. The cleaved compound was analyzed by LC-MS and the observed mass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phase peptide synthesis methods.

The solid support bound compound 143 was synthesized using standard solid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled and the solid support was filtered. The filtrate was concentrated and the residue dissolved in water and purified by HPLC on a strong anion exchange column. The fractions containing full length compound 144 were pooled together and desalted. The resulting GalNAc₄-11 conjugated oligomeric compound was analyzed by LC-MS and the observed mass was consistent with structure.

The GalNAc4 cluster portion of the conjugate group GalNAc₄-11 (GalNAc₄-11_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-1 l_(a)-CM) is shown below:

Example 51 Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was quenched by pouring into solution of saturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organic layer was separated, washed with brine, dried over MgSO₄, filtered, and was concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂to yield Compound 112 (16.53 g, 63%). LCMS and ¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in CH₂Cl₂, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL). N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15 minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added and stirred for 2 h under nitrogen atmosphere. The reaction was diluted with EtOAc and washed with saturated aqueous NaHCO₃ and brine. The organics phase was separated, dried (MgSO₄), filtered, and concentrated to an orange syrup. The crude material was purified by column chromatography 2-5% MeOH in CH₂Cl₂to yield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent with the expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (350 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with desired product. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The reaction mixture was allowed to stir for 15 minutes at room temperature under nitrogen. A solution of compound 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of the reaction was adjusted to pH=9-10 by addition of DIEA (if necessary). The reaction was allowed to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with EtOAc (100 mL), and washed with aqueous saturated aqueous NaHCO₃, followed by brine. The organic phase was separated, dried over MgSO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR were consistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the desired product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76 mmol) were added. The reaction mixture turned magenta upon contact, and gradually turned orange over the next 30 minutes. Progress of reaction was monitored by TLC and LCMS. Upon completion (formation of the PFP ester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction was adjusted to pH=9-10 by addition of N,N-Diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for 30 min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. The organic phase separated, dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g, 55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The reaction mixture was purged by bubbling a stream of argon thru the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (35 mg). Hydrogen gas was bubbled thru the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 153 (0.33 g, quantitative). The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL) with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL, 0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reaction mixture was stirred under nitrogen for ˜30 min. The reaction mixture turned magenta upon contact, and gradually turned orange. The pH of the reaction mixture was maintained at pH=9-10 by adding more N,-Diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), and washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10% MeOH in CH₂Cl₂to yield Compound 154 (0.29 g, 79%). LCMS and ¹H NMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52 Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL). Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbon powder, wet) was added. The reaction mixture was stirred at room temperature under hydrogen for 18 h. The reaction mixture was filtered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined filtrate was washed and concentrated to dryness. The residue was purified by silica gel column chromatography and eluted with 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL). HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81 mmol) were added and the reaction mixture was stirred at room temperature for 5 minutes. To this a solution of compound 47 (2.96 g, 7.04 mmol) was added. The reaction was stirred at room temperature for 8 h. The reaction mixture was poured into a saturated NaHCO₃ aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na₂SO₄), filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reduced pressure. The dried compound was dissolved in anhydrous DMF (50 mL). To this 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL, 3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite (3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred t under an argon atmosphere for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL). The reaction mixture was washed with saturated NaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90% ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and ³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, was prepared using standard oligonucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous ammonia yielded compound 160. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9 (GalNAc₃-9_(a)-CM) is shown below:

Example 53 Alternate Procedure for Preparation of Compound 18 (GalNAc₃-1a and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Boc protected diamino propane (1 eq) to provide alcohol 162a or 162b. When unprotected propanediamine was used for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected using CbzCl to provide 162b as a white solid after purification by column chromatography. Alcohol 162b was further reacted with compound 4 in the presence of TMSOTf to provide 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine (3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18. The above method greatly facilitates purification of intermediates and minimizes the formation of byproducts which are formed using the procedure described in Example 4.

Example 54 Alternate Procedure for Preparation of Compound 18 (GalNAc₃-1a and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedure outlined in example 53 above and reacted with mono-Boc protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or trifluoroacetic acid to provide the triamine which was reacted with the PFP activated acid 166 in the presence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the corresponding acid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF. The precursor acid in turn was prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by reaction with 1,6-hexanediol (or 1,5-pentanediol or other diol for other n values) (2-4 eq) and TMSOTf using conditions described previously in example 47.

Example 55 Dose-Dependent Study of Oligonucleotides Comprising Either a 3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃ conjugate groups was attached at either the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 39 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′) Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 none 2304 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -9 _(a) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 2304 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 2304 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-9 was shown previously in Example 52. The structure of GalNAc₃-3 was shown previously in Example 39. The structure of GalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664078, 661161, 665001 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 and ISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate at the 3′ terminus was essentially equipotent compared to ISIS 655861, which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′ conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potency compared to the 3′ conjugated antisense oligonucleotides (ISIS 655861 and ISIS 664078).

TABLE 40 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100 353382 3 88 none 10 68 30 36 655861 0.5 98 GalNAc₃-1 (3′) 1.5 76 5 31 15 20 664078 0.5 88 GalNAc₃-9 (3′) 1.5 85 5 46 15 20 661161 0.5 92 GalNAc₃-3 (5′) 1.5 59 5 19 15 11 665001 0.5 100 GalNAc₃-8 (5′) 1.5 73 5 29 15 13

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 41 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.2 30 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNAc₃-1 (3′) 1.5 23 48 0.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3 GalNAc₃-9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2 661161 0.5 101 162 0.1 34.17 GalNAc₃-3 (5′) 15 g 42 100 0.1 33.37  5 g 23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32 GalNAc₃-8 (5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56 Dose-Dependent Study of Oligonucleotides Comprising Either a 3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety) except for ISIS 655861 which had the GalNAc₃ conjugate group attached at the 3′ terminus.

TABLE 42 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′) Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 no conjugate 2304 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS 664507 GalNAc ₃ -2 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -2 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 2304 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -6 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-2_(a) was shown previously in Example 37. The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-5_(a) was shown previously in Example 49. The structure of GalNAc₃-6_(a) was shown previously in Example 51. The structure of GalNAc₃-7_(a) was shown previously in Example 48. The structure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the conjugated antisense oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). The 5′ conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3′ conjugated antisense oligonucleotide.

TABLE 43 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNAc₃-1 (3′) 1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNAc₃-2 (5′) 1.5 73.2 5 31.3 15 10.8 661161 0.5 90.7 GalNAc₃-3 (5′) 1.5 67.6 5 24.3 15 11.5 666224 0.5 96.1 GalNAc₃-5 (5′) 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5 GalNAc₃-6 (5′) 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc₃-7 (5′) 1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0 GalNAc₃-10 (5′) 1.5 65.8 5 26.0 15 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.

TABLE 44 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 54 0.1 28 655861 0.5 25 71 0.2 34 GalNAc₃-1 (3′) 1.5 28 60 0.2 26 5 26 63 0.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNAc₃-2 (5′) 1.5 24 49 0.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNAc₃-3 (5′) 1.5 g 37 74 0.2 25   5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5 34 48 0.2 21 GalNAc₃-5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 32 49 0.1 26 666961 0.5 17 63 0.2 26 GalNAc₃-6 (5′) 1.5 23 68 0.2 26 5 25 66 0.2 26 15 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc₃-7 (5′) 1.5 30 55 0.2 24 5 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27 GalNAc₃-10 (5′) 1.5 23 59 0.2 24 5 45 70 0.2 26 15 21 57 0.2 24

Example 57 Duration of Action Study of Oligonucleotides Comprising a 3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitored over the course of 42 days for ApoC-III and plasma triglycerides (Plasma TG) levels. The study was performed using 3 transgenic mice that express human APOC-III in each group.

TABLE 45 Modified ASO targeting ApoC III SEQ ID ASO Sequence (5′ to 3′) Linkages No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 2296 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds) PS 2297 647535 A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds) PO/PS 2297 647536 A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

TABLE 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (% Saline on Day 1) Day Day Day Day Day ASO Dose Target 3 7 14 35 42 Saline  0 mg/kg ApoC-III 98 100 100 95 116 ISIS 30 mg/kg ApoC-III 28 30 41 65 74 304801 ISIS 10 mg/kg ApoC-III 16 19 25 74 94 647535 ISIS 10 mg/kg ApoC-III 18 16 17 35 51 647536 Saline  0 mg/kg Plasma TG 121 130 123 105 109 ISIS 30 mg/kg Plasma TG 34 37 50 69 69 304801 ISIS 10 mg/kg Plasma TG 18 14 24 18 71 647535 ISIS 10 mg/kg Plasma TG 21 19 15 32 35 647536

As can be seen in the table above the duration of action increased with addition of the 3′-conjugate group compared to the unconjugated oligonucleotide. There was a further increase in the duration of action for the conjugated mixed PO/PS oligonucleotide 647536 as compared to the conjugated full PS oligonucleotide 647535.

Example 58 Dose-Dependent Study of Oligonucleotides Comprising a 3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc₄-11) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 663748 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-1 and GalNAc₄-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). The two conjugated oligonucleotides, GalNAc₃-1 and GalNAc₄-11, were equipotent.

TABLE 47 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence (5′ to 3′) Dose mg/kg control No. Saline 100 ISIS 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.6 73.45 2298 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS 651900 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 62.75 2299 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1 _(a) 0.6 29.14 2 8.61 6 5.62 ISIS 663748 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 63.99 2299 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₄ -11 _(a) 0.6 33.53 2 7.58 6 5.52

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 48 below.

TABLE 48 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN Conjugate Saline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 48 0.1 39 651900 0.2 32 115 0.2 39 GalNAc₃-1 (3′) 0.6 33 61 0.1 35 2 30 50 0.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNAc₄-11 (3′) 0.6 34 60 0.1 35 2 44 62 0.1 36 6 38 71 0.1 33

Example 59 Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of FXI in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugate groups was attached at the 3′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 49 Modified ASOs targeting FXI SEQ ID ASO Sequence (5′ to 3′) Linkages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 2307 404071 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 2308 656172 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) PO/PS 2308 656173 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously twice a week for 3 weeks at the dosage shown below with ISIS 404071, 656172, 656173 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Plasma FXI protein levels were also measured using ELISA. FXI mRNA levels were determined relative to total RNA (using RIBOGREEN®), prior to normalization to PBS-treated control. The results below are presented as the average percent of FXI mRNA levels for each treatment group. The data was normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

TABLE 50 Factor XI mRNA (% Saline) Dose ASO mg/kg % Control Conjugate Linkages Saline 100 none ISIS 3 92 none PS 404071 10 40 30 15 ISIS 0.7 74 GalNAc₃-1 PS 656172 2 33 6 9 ISIS 0.7 49 GalNAc₃-1 PO/PS 656173 2 22 6 1

As illustrated in Table 50, treatment with antisense oligonucleotides lowered FXI mRNA levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a Factor XI protein (% Saline) Dose Protein ASO mg/kg (% Control) Conjugate Linkages Saline 100 none ISIS 3 127 none PS 404071 10 32 30 3 ISIS 0.7 70 GalNAc₃-1 PS 656172 2 23 6 1 ISIS 0.7 45 GalNAc₃-1 PO/PS 656173 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 51 ISIS Dosage Total Total No. mg/kg ALT AST Albumin Bilirubin CRE BUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.1 0.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.2 0.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNAc₃-1 (3′) 2 33.0 51.8 2.9 0.2 0.2 22.0 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.5 3.0 0.2 0.2 24.9 GalNAc₃-1 (3′) 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0 101.8 3.3 0.2 0.2 22.7

Example 60 Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 52 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′) Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 none 2304 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1a ISIS 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1a ISIS 661161 GalNAc ₃ -3 _(a-o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -3 2306 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃ -8 _(a-o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -8 2306 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -9 _(a) ISIS 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -6 2306 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664507 GalNAc ₃ -2 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -2 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. The structure of GalNAc₃-3a was shown previously in Example 39. The structure of GalNAc₃-8a was shown previously in Example 47. The structure of GalNAc₃-9a was shown previously in Example 52. The structure of GalNAc₃-6a was shown previously in Example 51. The structure of GalNAc₃-2a was shown previously in Example 37. The structure of GalNAc₃-10a was shown previously in Example 46. The structure of GalNAc₃-5a was shown previously in Example 49. The structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modified oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the SRB-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results are presented in Table 53. The results show that, under free uptake conditions in which no reagents or electroporation techniques are used to artificially promote entry of the oligonucleotides into cells, the oligonucleotides comprising a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.

TABLE 53 IC₅₀ Internucleoside SEQ ID ASO (nM) linkages Conjugate No. ISIS 353382 190^(a) PS none 2304 ISIS 655861  11^(a) PS GalNAc₃-1 2305 ISIS 655862  3 PO/PS GalNAc₃-1 2305 ISIS 661161  15^(a) PS GalNAc₃-3 2306 ISIS 665001  20 PS GalNAc₃-8 2306 ISIS 664078  55 PS GalNAc₃-9 2305 ISIS 666961  22^(a) PS GalNAc₃-6 2306 ISIS 664507  30 PS GalNAc₃-2 2306 ISIS 666881  30 PS GalNAc₃-10 2306 ISIS 666224  30^(a) PS GalNAc₃-5 2306 ISIS 666981  40 PS GalNAc₃-7 2306 ^(a)Average of multiple runs.

Example 61 Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared by addition of benzyl (perfluorophenyl) glutarate to compound 171. The benzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound 175, comprising a GalNAc₃-12 conjugate group, was prepared from compound 174 using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12 (GalNAc₃-12_(a)-CM-) is shown below:

Example 62 Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example 2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, was prepared from compound 177 using the general procedures illustrated in Example 49. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-13 (GalNAc₃-13_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63 Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a GalNAc₃-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-14 (GalNAc₃-14_(a)-CM-) is shown below:

Example 64 Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared using the general procedure shown in Example 31. Oligomeric compound 197, comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194 and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15 (GalNAc₃-15_(a)-CM-) is shown below:

Example 65 Dose-Dependent Study of Oligonucleotides Comprising a 5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54 Modified ASOs targeting SRB-1 ISIS SEQ No. Sequences (5′ to 3′) Conjugate ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) none 2304 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3 2306 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671144 GalNAc ₃ -12 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-12 2306 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670061 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-13 2306 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671261 GalNAc ₃ -14 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-14 2306 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 671262 GalNAc ₃ -15 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-15 2306 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-12a was shown previously in Example 61. The structure of GalNAc₃-13a was shown previously in Example 62. The structure of GalNAc₃-14a was shown previously in Example 63. The structure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57b16 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once or twice at the dosage shown below with ISIS 353382, 661161, 671144, 670061, 671261, 671262, or with saline. Mice that were dosed twice received the second dose three days after the first dose. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. No significant differences in target knockdown were observed between animals that received a single dose and animals that received two doses (see ISIS 353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5 mg/kg). The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

TABLE 55 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA ED₅₀ No. (mg/kg) (% Saline) (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.0 22.4 none 10 69.2 30 34.2 2 × 15 36.0 661161 0.5 87.4 2.2 GalNAc₃-3 1.5 59.0 5 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.5 76.1 5 32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 15 13.3 671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5 109.4 9.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56 Total Bili- ISIS Dosage ALT AST rubin BUN No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.2 36 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15  22 59 0.2 33 661161 0.5 39 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.2 28 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃-12 1.5 26 55 0.2 28 5 48 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃-13 1.5 24 45 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33 GalNAc₃-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 24 51 0.2 29 GalNAc₃-15 1.5 32 62 0.1 31 5 30 76 0.2 32 15 31 64 0.1 32

Example 66 Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃ Cluster

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 57 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d) 2309 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 670700 GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 670701 GalNAc ₃ -3 _(a) - _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 2309 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 671165 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising various cleavable moieties all demonstrated similar potencies.

TABLE 58 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8 GalNAc₃-3a A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3a T_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a A_(e) 1.5 63.3 5 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a T_(e) 1.5 59.2 5 35.8 15 17.7 671165 0.5 76.4 GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 59 below.

TABLE 59 Total Bili- rubin BUN ISIS Dosage ALT AST (mg/ (mg/ GalNAc₃ No. (mg/kg) (U/L) (U/L) dL) dL) Cluster CM Saline n/a 24 64 0.2 31 n/a n/a 661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 55 0.2 28 15 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 33 58 0.2 32 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27 GalNAc₃-3a A_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 670701 0.5 30 77 0.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 31 82 0.2 24 671165 0.5 44 84 0.2 26 GalNAc₃-13a A_(d) 1.5 47 71 0.1 24 5 33 91 0.2 26 15 33 56 0.2 29

Example 67 Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16 (GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.The structure of GalNAc₃-16 (GalNAc₃-16_(a)-CM-) is shown below:

Example 68 Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-17 (GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-17 (GalNAc₃-17_(a)-CM-) is shown below:

Example 69 Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-18 (GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-18 (GalNAc₃-18_(a)-CM-) is shown below:

Example 70 Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, was prepared from compound 64 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71 Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which was prepared by adding triflic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80° C., then lowered to rt. Oligomeric compound 210, comprising a GalNAc₃-20 conjugate group, was prepared from compound 208 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-20 (GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20 (GalNAc₃-20_(a)-CM-) is shown below:

Example 72 Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215, comprising a GalNAc₃-21 conjugate group, was prepared from compound 213 using the general procedures illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-2 1_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21 (GalNAc₃-2 l_(a)-CM-) is shown below:

Example 73 Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugate group, is prepared from compound 220 using the general procedure illustrated in Example 52. The GalNAc₃ cluster portion of the conjugate group GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74 Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugate groups was attached at the 5′ terminus of the respective oligonucleotide.

TABLE 60 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) n/a n/a 2304 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 2304 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675441 GalNAc ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(dsT) _(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675442 GalNAc ₃ -18 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

In all tables, capital letters indicate the nucleobase for each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. The structure of GalNAc₃-17a was shown previously in Example 68, and the structure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 60 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc conjugate showed similar potencies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 61 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38 n/a n/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a A_(d) 1.5 75.96 5 30.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a PO 1.5 57.88 5 21.22 15 16.49 675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49 675442 0.5 95.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 62 below.

TABLE 62 Total Bili- rubin BUN ISIS Dosage ALT AST (mg/ (mg/ GalNAc₃ No. (mg/kg) (U/L) (U/L) dL) dL) Cluster CM Saline n/a 26 59 0.16 42 n/a n/a 353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34 661161 0.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.15 39 15 32 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 48 0.12 32 5 19 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36 GalNAc₃-17a A_(d) 1.5 60 95 0.16 34 5 27 75 0.14 37 15 24 61 0.14 36 675442 0.5 26 65 0.15 37 GalNAc₃-18a A_(d) 1.5 25 64 0.15 43 5 27 69 0.15 37 15 30 84 0.14 37

Example 75 Pharmacokinetic Analysis of Oligonucleotides Comprising a 5′-Conjugate Group

The PK of the ASOs in Tables 54, 57 and 60 above was evaluated using liver samples that were obtained following the treatment procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using standard protocols and analyzed by IP-HPLC-MS alongside an internal standard. The combined tissue level (μg/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate (“parent,” which is Isis No. 353382 in this case) was measured using the appropriate extracted ion chromatograms (EIC).

TABLE 63 PK Analysis in Liver Total Parent ASO Tissue Level Tissue Level ISIS Dosage by UV by EIC GalNAc₃ No. (mg/kg) (μg/g) (μg/g) Cluster CM 353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7 GalNAc₃-3a A_(d) 15 63.2 44.1 671144 5 20.5 19.2 GalNAc₃-12a A_(d) 15 48.6 41.5 670061 5 31.6 28.0 GalNAc₃-13a A_(d) 15 67.6 55.5 671261 5 19.8 16.8 GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4 GalNAc₃-15a A_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a T_(d) 15 31.5 22.5 670700 5 19.3 10.9 GalNAc₃-3a A_(e) 15 38.1 20.0 670701 5 21.8 8.8 GalNAc₃-3a T_(e) 15 35.2 16.1 671165 5 27.1 26.5 GalNAc₃-13a A_(d) 15 48.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a PO 15 52.6 37.6 675441 5 25.4 19.0 GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7 GalNAc₃-18a A_(d) 15 39.6 29.0

The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a GalNAc₃ conjugate group than of the parent oligonucleotide that does not comprise a GalNAc₃ conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking into consideration the differences in dosing between the oligonucleotides with and without a GalNAc₃ conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a GalNAc₃ conjugate group was metabolized to the parent compound, indicating that the GalNAc₃ conjugate groups were cleaved from the oligonucleotides.

Example 76 Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500 mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in EtOAc and washed with water, sat. NaHCO₃, brine, and dried over Na₂SO₄. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with EtOAc/hexanes (1:1) followed by 10% methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMR were consistent with the structure. 10 g (32.86 mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17 hours. The reaction mixture was then poured onto water, and extracted with EtOAc. The organic layer was washed with water three times and dried over Na₂SO₄. The organic layer was concentrated to dryness to give 5.3g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves (5g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) under an inert atmosphere. After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat. NaHCO₃, water, brine, and dried over Na₂SO₄. The organic layer was concentrated to dryness and purified by column chromatography, eluted with a gradient of 2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMR were consistent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman's catalyst. After 8 hours, the catalyst was removed by filtration and the solvents removed to dryness to give compound 226. LCMS and NMR were consistent with the structure.

In order to generate compound 227, a solution of nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base (10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise with pentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the reaction was poured onto ice water and extracted with EtOAc. The organic layer was washed with water, brine, and dried over Na₂SO₄. The organic layer was concentrated to dryness and then recrystallized from heptane to give compound 227 as a white solid. LCMS and NMR were consistent with the structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 mL) for 2 hours. The reaction was then evaporated to dryness and purified by column chromatography, eluting with a gradient of 2 to10% methanol in dichloromethane to give compound 228. LCMS and NMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney Nickel (about 2g wet) in ethanol (100 mL) in an atmosphere of hydrogen. After 12 hours, the catalyst was removed by filtration and the organic layer was evaporated to a solid that was used directly in the next step. LCMS and NMR were consistent with the structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 μL 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removed under reduced pressure at 65° C. to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. NaHCO₃, brine, and dried over Na₂SO₄. After evaporation of the organic layer, the compound was purified by column chromatography and eluted with a gradient of 2 to 20% methanol in dichloromethane to give the coupled product. LCMS and NMR were consistent with the structure. The benzyl ester was deprotected with Pearlman's catalyst under a hydrogen atmosphere for 1 hour. The catalyst was them removed by filtration and the solvents removed to dryness to give the acid. LCMS and NMR were consistent with the structure. The acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was added and the reaction was purged with argon. Pentaflourotriflouro acetate (46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The color of the reaction changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The reaction was allowed to stir at room temperature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced pressure (rotovap) at 70° C. The residue was diluted with DCM and washed with IN NaHSO₄, brine, saturated sodium bicarbonate and brine again. The organics were dried over Na₂SO₄, filtered, and were concentrated to dryness to give 225 mg of compound 229 as a brittle yellow foam. LCMS and NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, was prepared from compound 229 using the general procedure illustrated in Example 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugate group (GalNAc₃-23_(a)) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc₃-23 (GalNAc₃-23_(a)-CM) is shown below:

Example 77 Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 64 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 2304 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 673502 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 677844 GalNAc ₃ -9 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 677843 GalNAc ₃ -23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d) 2306 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-1a A_(d) 2305 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-19a A_(d) 2305 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-20a A_(d) 2305 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-9a was shown in Example 52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown in Example 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a) was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 64 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65 SRB-1 mRNA (% Saline) ISIS Dosage SRB-1 mRNA GalNAc₃ No. (mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 89.18 GalNAc₃-3a A_(d) 1.5 77.02 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3a PO 1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75 GalNAc₃-10a A_(d) 1.5 41.05 5 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a A_(d) 1.5 93.04 5 40.77 15 16.95 677843 0.5 102.28 GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 15 13.26 655861 0.5 79.72 GalNAc₃-1a A_(d) 1.5 55.48 5 26.99 15 17.58 677841 0.5 67.43 GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 677842 0.5 64.13 GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN were also evaluated. Changes in body weights were evaluated, with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.

TABLE 66 Total Bili- rubin BUN ISIS Dosage ALT AST (mg/ (mg/ GalNAc₃ No. (mg/kg) (U/L) (U/L) dL) dL) Cluster CM Saline n/a 21 45 0.13 34 n/a n/a 661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 59 0.13 37 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 26 68 0.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34 GalNAc₃-10a A_(d) 1.5 20 46 0.17 32 5 24 45 0.12 31 15 24 47 0.13 34 677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.13 35 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃-23a A_(d) 1.5 25 54 0.13 34 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33 GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30 677841 0.5 32 54 0.13 34 GalNAc₃-19a A_(d) 1.5 24 56 0.14 34 5 23 92 0.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃-20a A_(d) 1.5 24 57 0.14 34 5 41 62 0.15 35 15 24 37 0.14 32

Example 78 Antisense Inhibition In Vivo by Oligonucleotides Targeting Angiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.

TABLE 67 Modified ASOs targeting AGT ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 552668 ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 2310 G_(es)A_(es)T_(e) 669509 ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es) GalNAc₃-1_(a) A_(d) 2311 G_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 67 or with PBS. Each treatment group consisted of 4 animals. The rats were sacrificed 72 hours following the final dose. AGT liver mRNA levels were measured using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. AGT plasma protein levels were measured using the Total Angiotensinogen ELISA (Catalog #JP27412, IBL International, Toronto, ON) with plasma diluted 1:20,000. The results below are presented as the average percent of AGT mRNA levels in liver or AGT protein levels in plasma for each treatment group, normalized to the PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotides lowered AGT liver mRNA and plasma protein levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 68 AGT liver mRNA and plasma protein levels AGT liver AGT plasma ISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 8 11 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in plasma and body weights were also measured at time of sacrifice using standard protocols. The results are shown in Table 69 below.

TABLE 69 Liver transaminase levels and rat body weights ISIS Dosage ALT AST Body Weight GalNAc₃ No. (mg/kg) (U/L) (U/L) (% of baseline) Cluster CM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93 194 30 59 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃-1a A_(d) 1 51 93 192 3 48 85 189 10 56 95 189

Example 79 Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action in mice.

TABLE 70 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 2296 T_(es)A_(es)T_(e) 647535 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1a A_(d) 2297 T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663083 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 2312 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449 GalNAc ₃ -7 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 2312 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 2312 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451 GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 2312 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six to eight week old transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 70 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results below are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels, showing that the oligonucleotides comprising a GalNAc conjugate group exhibited a longer duration of action than the parent oligonucleotide without a conjugate group (ISIS 304801) even though the dosage of the parent was three times the dosage of the oligonucleotides comprising a GalNAc conjugate group.

TABLE 71 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point Triglyc- APOC-III (days erides protein ISIS Dosage post- (% base- (% base- GalNAc₃ No. (mg/kg) dose) line) line) Cluster CM PBS n/a 3 97 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 42 91 105 304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 68 70 42 75 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 41 41 28 42 62 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 28 23 14 25 27 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26 GalNAc₃-7a A_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99 674450 10 3 33 30 GalNAc₃-10a A_(d) 7 35 34 14 31 34 21 44 44 28 56 61 35 68 70 42 83 95 674451 10 3 35 33 GalNAc₃-13a A_(d) 7 24 32 14 40 34 21 48 48 28 54 67 35 65 75 42 74 97

Example 80 Antisense Inhibition In Vivo by Oligonucleotides Targeting Alpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 below were tested in a study for dose-dependent inhibition of A1AT in mice.

TABLE 72 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 2313 G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1a A_(d) 2314 G_(es)G_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 678381 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds) GalNAc₃-3a A_(d) 2315 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678382 GalNAc ₃ -7 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds) GalNAc₃-7a A_(d) 2315 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678383 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-10a A_(d) 2315 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678384 GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-13a A_(d) 2315 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. A1AT liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. A1AT plasma protein levels were determined using the Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco, Salem, N.H.). The results below are presented as the average percent of A1AT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.

As illustrated in Table 73, treatment with antisense oligonucleotides lowered A1AT liver mRNA and A1AT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 476366).

TABLE 73 A1AT liver mRNA and plasma protein levels A1AT liver A1AT plasma ISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS) Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 30 38 656326 0.6 99 90 GalNAc₃-1a A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6 105 90 GalNAc₃-3a A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79 GalNAc₃-7a A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc₃-10a A_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc₃-13a A_(d) 2 65 59 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time of sacrifice using standard protocols. Body weights and organ weights were also measured. The results are shown in Table 74 below. Body weight is shown as % relative to baseline. Organ weights are shown as % of body weight relative to the PBS control group.

TABLE 74 Body Liver Kidney Spleen ISIS Dosage ALT AST BUN weight weight weight weight No. (mg/kg) (U/L) (U/L) (mg/dL) (% baseline) (Rel % BW) (Rel % BW) (Rel % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 34 68 35 116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123 656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 32 67 39 125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.6 30 67 38 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105 105 18 36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 61 32 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81 Duration Of Action In Vivo of Oligonucleotides Targeting A1AT Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 were tested in a single dose study for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline and at 5, 12, 19, and 25 days following the dose. Plasma A1AT protein levels were measured via ELISA (see Example 80). The results below are presented as the average percent of plasma A1AT protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent lacking a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382, 678383, and 678384) were generally even more potent with even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656326).

TABLE 75 Plasma A1AT protein levels in mice Time point ISIS Dosage (days post- A1AT (% GalNAc₃ No. (mg/kg) dose) baseline) Cluster CM PBS n/a 5 93 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 62 25 77 656326 18 5 33 GalNAc₃-1a A_(d) 12 36 19 51 25 72 678381 18 5 21 GalNAc₃-3a A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a A_(d) 12 21 19 39 25 60 678383 18 5 24 GalNAc₃-10a A_(d) 12 21 19 45 25 73 678384 18 5 29 GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82 Antisense Inhibition In Vitro by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250 nM in Williams E medium and cells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16 hours following oligonucleotide addition, and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. IC50 values were determined using Prism 4 software (GraphPad). The results show that oligonucleotides comprising a variety of different GalNAc conjugate groups and a variety of different cleavable moieties are significantly more potent in an in vitro free uptake experiment than the parent oligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and 666841).

TABLE 76 Inhibition of SRB-1 expression in vitro ISIS GalNAc IC₅₀ SEQ No. Sequence (5′ to 3′) Linkages cluster CM (nM) ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 250 2304 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 1_(a) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 661162 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 664078 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 20 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -9 _(a) 9_(a) 665001 GalNAc ₃ -8 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 70 2306 ^(m)C_(ds)-A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 8_(a) 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 80 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 5_(a) 666841 G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PO/PS n/a n/a >250 2304 ^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 666881 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 10_(a) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) PS GalNAc₃- PO 9 2304 A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666924 GalNAc ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- T_(d) 15 2309 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 150 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 6_(a) 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 7_(a) 670061 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 13_(a) 670699 GalNAc ₃ -3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(d) 15 2309 ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 670700 GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(e) 30 2306 ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T 3_(a) 670701 GalNAc ₃ -3 _(a) - _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(e) 25 2306 ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 671144 GalNAc ₃ -12 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 12_(a) 671165 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 2306 ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T 13_(a) 671261 GalNAc ₃ -14 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) >250 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 14_(a) 671262 GalNAc ₃ -15 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) >250 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 15_(a) 673501 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 30 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 7_(a) 673502 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 10_(a) 675441 GalNAc ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 17_(a) 675442 GalNAc ₃ -18 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 18_(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a) 19_(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 30 2305 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a) 20_(a) 677843 GalNAc ₃ -23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 2306 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 23_(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown in Example 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-8_(a) was shown in Example 47, GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) was shown in Example 62, GalNAc₃-14_(a) was shown in Example 63, GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown in Example 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) was shown in Example 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a) was shown in Example 76.

Example 83 Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor XI in mice.

TABLE 77 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQ No. Sequence (5′ to 3′) cluster CM ID No. 404071 T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 2307 A_(es)G_(es)G_(e) 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo) GalNAc₃-1_(a) A_(d) 2308 A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663086 GalNAc ₃ -3 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3_(a) A_(d) 2316 T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃ -7 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-7_(a) A_(d) 2316 T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃ -10 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-10_(a) A_(d) 2316 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃ -13 _(a) - _(o′) A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-13_(a) A_(d) 2316 T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a) was shown in Example 62.

Treatment

Six to eight week old mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final dose. Factor XI liver mRNA levels were measured using real-time PCR and normalized to cyclophilin according to standard protocols. Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each treatment group, normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotides lowered Factor XI liver mRNA in a dose-dependent manner. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubin levels Factor XI ISIS Dosage mRNA ALT AST BUN Bilirubin GalNAc₃ SEQ No. (mg/kg) (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a 100 63 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 2307 10 33 49 53 23 0.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 2308 2 9 36 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16 GalNAc₃-3a 2316 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 20 0.14 GalNAc₃-7a 2316 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.7 39 43 54 21 0.16 GalNAc₃-10a 2316 2 5 38 55 22 0.17 6 2 25 38 20 0.14 678349 0.7 34 39 46 20 0.16 GalNAc₃-13a 2316 2 8 43 63 21 0.14 6 2 28 41 20 0.14

Example 84 Duration of Action In Vivo of Oligonucleotides Targeting Factor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.

Treatment

Six to eight week old mice were each injected subcutaneously once with an oligonucleotide listed in Table 77 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to determine baseline and at 3, 10, and 17 days following the dose. Plasma Factor XI protein levels were measured by ELISA using Factor XI capture and biotinylated detection antibodies from R & D Systems, Minneapolis, Minn. (catalog #AF2460 and #BAF2460, respectively) and the OptEIA Reagent Set B (Catalog #550534, BD Biosciences, San Jose, Calif.). The results below are presented as the average percent of plasma Factor XI protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79 Plasma Factor XI protein levels in mice Time point Factor (days XI ISIS Dosage post- (% base- GalNAc₃ SEQ No. (mg/kg) dose) line) Cluster CM ID No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/a n/a 2307 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 2308 10 3 17 21 663086 6 3 1 GalNAc₃-3a A_(d) 2316 10 2 17 9 678347 6 3 1 GalNAc₃-7a A_(d) 2316 10 1 17 8 678348 6 3 1 GalNAc₃-10a A_(d) 2316 10 1 17 6 678349 6 3 1 GalNAc₃-13a A_(d) 2316 10 1 17 5

Example 85 Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 76 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of liver SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80 SRB-1 mRNA in liver Dosage SRB-1 mRNA (% GalNAc₃ ISIS No. (mg/kg) Saline) Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94 GalNAc₃-1a A_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a A_(d) 0.3 107 1 68 3 26 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 666981 0.1 120 GalNAc₃-7a A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13a A_(d) 0.3 89 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 81 SRB-1 mRNA in liver SRB-1 mRNA (% GalNAc₃ ISIS No. Dosage (mg/kg) Saline) Cluster CM 661161 0.1 107 GalNAc₃-3a A_(d) 0.3 95 1 53 3 18 677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights were also measured using standard protocols. Average values for each treatment group are shown in Table 82 below.

TABLE 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight GalNAc₃ No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster CM Saline n/a 19 39 0.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d) 0.3 29 56 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.1 35 83 0.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22 116 3 29 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d) 0.3 49 82 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.1 21 41 0.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22 111 3 77 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d) 0.3 20 57 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.1 20 38 0.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.15 21 119 3 41 57 0.14 23 118

Example 86 Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in the tables below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Tail bleeds were performed at various time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 84-87. After the animals were sacrificed, plasma ALT, AST, and human TTR levels were measured, as were body weights, organ weights, and liver human TTR mRNA levels. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, Calif.). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Tables 84-87 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. Body weights are the average percent weight change from baseline until sacrifice for each individual treatment group. Organ weights shown are normalized to the animal's body weight, and the average normalized organ weight for each treatment group is then presented relative to the average normalized organ weight for the PBS group.

In Tables 84-87, “BL” indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Tables 84 and 85, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the oligonucleotide comprising a GalNAc conjugate and full PS linkages.

TABLE 83 Oligonucleotides targeting human TTR GalNAc SEQ Isis No. Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(es) PS n/a n/a 2317 T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es)mC_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(es) PS GalNAc₃-1a A_(d) 2318 T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo)

682883

T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) PS/PO GalNAc₃-3a PO 2317 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682884

T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) PS/PO GalNAc₃-7a PO 2317 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682885

T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/PO GalNAc₃-10a PO 2317 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682886

T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/PO GalNAc₃-13a PO 2317 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684057 T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo) PS/PO GalNAc₃-19a A_(d) 2318 T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo)

The legend for Table 85 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-l0_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62. The structure of GalNAc₃-19_(a) was shown in Example 70.

TABLE 84 Antisense inhibition of human TTR in vivo Plasma TTR TTR Isis Dosage mRNA protein GalNAc SEQ No. (mg/kg) (% PBS) (% PBS) cluster CM ID No. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 2317 20 48 65 60 18 28 660261 0.6 113 87 GalNAc3-1a A_(d) 2318 2 40 56 6 20 27 20 9 11

TABLE 85 Antisense inhibition of human TTR in vivo Plasma TTR protein (% PBS at BL) Isis Dosage TTR mRNA Day 17 GalNAc SEQ No. (mg/kg) (% PBS) BL Day 3 Day 10 (After sac) cluster CM ID No. PBS n/a 100 100 96 90 114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 2317 20 43 102 66 61 58 60 24 92 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃-3a PO 2317 2 18 75 38 23 23 6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃-7a PO 2317 2 19 76 44 25 23 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃-10a PO 2317 2 22 93 58 32 32 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃-13a PO 2317 2 21 89 50 31 30 6 18 102 41 24 27 684057 0.6 53 80 69 56 62 GalNAc₃-19a A_(d) 2318 2 21 92 55 34 30 6 11 82 50 18 13

TABLE 86 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) Isis Dosage Day Day Day Day Day Day Body Liver Spleen Kidney SEQ No. (mg/kg) BL 3 10 17 BL 3 10 17 (% BL) (% PBS) (% PBS) (% PBS) ID No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a 420915 6 34 33 27 21 64 59 73 47 115 99 89 91 2317 20 34 30 28 19 64 54 56 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 660261 0.6 33 38 28 26 70 71 63 59 111 96 99 92 2318 2 29 32 31 34 61 60 68 61 118 100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 64 54 68 95 114 101 106 92

TABLE 87 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) Isis Dosage Day Day Day Day Day Day Body Liver Spleen Kidney SEQ No. (mg/kg) BL 3 10 17 BL 3 10 17 (% BL) (% PBS) (% PBS) (% PBS) ID No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a 420915 6 32 30 34 34 61 71 72 66 102 103 102 105 2317 20 41 34 37 33 80 76 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99 682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 2317 2 38 39 42 43 71 84 70 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 682884 0.6 33 32 35 34 70 74 75 67 101 100 130 99 2317 2 31 32 38 38 63 77 66 55 104 103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 26 37 35 63 63 77 59 100 109 109 112 2317 2 30 26 38 40 54 56 71 72 102 98 111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34 36 58 87 54 61 104 99 120 101 2317 2 27 26 34 36 51 55 55 69 103 91 105 92 6 40 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 56 51 51 69 104 99 110 102 2318 2 33 32 31 40 54 57 56 87 103 100 112 97 6 39 33 35 40 67 52 55 92 98 104 121 108

Example 87 Duration of Action In Vivo by Single Doses of Oligonucleotides Targeting TTR Comprising a GalNAc₃ Conjugate

ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS numbers 420915, 682883, and 682885 (see Table 83) were also tested in a single dose study for duration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 88 Plasma TTR protein levels Time TTR point (% ISIS Dosage (days post- base- GalNAc₃ SEQ No. (mg/kg) dose) line) Cluster CM ID No. 420915 100 3 30 n/a n/a 2317 7 23 10 35 17 53 24 75 39 100 660261 13.5 3 27 GalNAc3-1a A_(d) 2318 7 21 10 22 17 36 24 48 39 69

Treatment

Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 89 Plasma TTR protein levels Time TTR point (% ISIS Dosage (days post- base- GalNAc₃ SEQ No. (mg/kg) dose) line) Cluster CM ID No. 420915 100 3 48 n/a n/a 2317 7 48 10 48 17 66 31 80 682883 10.0 3 45 GalNAc₃-3a PO 2317 7 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO 2317 7 33 10 34 17 40 31 64

The results in Tables 88 and 89 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer duration of action than the parent oligonucleotide lacking a conjugate (ISIS 420915).

Example 88 Splicing Modulation In Vivo by Oligonucleotides Targeting SMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.

TABLE 90 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es) n/a n/a 2319 G_(e) 699819 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es) GalNAc₃-7a PO 2319 T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)G_(e) 699821 GalNAc ₃ -7 _(a) - _(o′)A_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(eo) ^(m)C_(eo)T_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)T_(eo)A_(eo) GalNAc₃-7a PO 2319 A_(eo)T_(eo)G_(eo) ^(m)C_(eo)T_(es)G_(es)G_(e) 700000 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es) GalNAc₃-1a A_(d) 2320 G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 703421 X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 2319 703422 GalNAc ₃ -7 _(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃-7b n/a 2319 The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X” indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.), and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the —NH—C₆—O portion of the linker, as shown below:

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide.

Treatment

Six week old transgenic mice that express human SMN were injected subcutaneously once with an oligonucleotide listed in Table 91 or with saline. Each treatment group consisted of 2 males and 2 females. The mice were sacrificed 3 days following the dose to determine the liver human SMN mRNA levels both with and without exon 7 using real-time PCR according to standard protocols. Total RNA was measured using Ribogreen reagent. The SMN mRNA levels were normalized to total mRNA, and further normalized to the averages for the saline treatment group. The resulting average ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table 91. The results show that fully modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in altering splicing in the liver than the parent oligonucleotides lacking a GlaNAc conjugate. Furthermore, this trend is maintained for multiple modification chemistries, including 2′-MOE and morpholino modified oligonucleotides.

TABLE 91 Effect of oligonucleotides targeting human SMN in vivo ISIS GalNAc₃ SEQ No. Dose (mg/kg) +Exon 7/−Exon 7 Cluster CM ID No. Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 2319 387954 288 5.00 n/a n/a 2319 699819 32 7.84 GalNAc₃-7a PO 2319 699821 32 7.22 GalNAc₃-7a PO 2319 700000 32 6.91 GalNAc₃-1a A_(d) 2320 703421 32 1.27 n/a n/a 2319 703422 32 4.12 GalNAc₃-7b n/a 2319

Example 89 Antisense Inhibition In Vivo by Oligonucleotides Targeting Apolipoprotein A (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No. Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds) n/a n/a 2321 T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo) GalNAc₃-7a PO 2321 T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds) G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) T_(ds)T_(eo) G_(eo)T_(es)T_(es) ^(m)C_(e) The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected subcutaneously once per week at a dosage shown below, for a total of six doses, with an oligonucleotide listed in Table 92 or with PBS. Each treatment group consisted of 3-4 animals. Tail bleeds were performed the day before the first dose and weekly following each dose to determine plasma Apo(a) protein levels. The mice were sacrificed two days following the final administration. Apo(a) liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma protein results in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were further normalized to the baseline (BL) value for the PBS group. Average absolute transaminase levels and body weights (% relative to baseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the oligonucleotides lowered Apo(a) liver mRNA and plasma protein levels in a dose-dependent manner. Furthermore, the oligonucleotide comprising the GalNAc conjugate was significantly more potent with a longer duration of action than the parent oligonucleotide lacking a GalNAc conjugate. As illustrated in Table 94, transaminase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.

TABLE 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a) mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 PBS n/a 100 100 120 119 113 88 121 97 494372 3 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 7 9 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 82 52 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 94 Body weight ISIS No. Dosage (mg/kg) ALT (U/L) AST (U/L) (% baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103 681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90 Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in Table 96 or with PBS. Each treatment group consisted of 4 animals. Prior to the first dose, a tail bleed was performed to determine plasma TTR protein levels at baseline (BL). The mice were sacrificed 72 hours following the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, Calif.). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Table 96 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. “BL” indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Table 96, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or deoxyadenosine cleavable moiety showed significant improvements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95 Oligonucleotides targeting human TTR GalNAc SEQ Isis No. Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es) ^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 2317 A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds) PS/PO GalNAc₃-3a PO 2317 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-3a A_(d) 2322 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682887 GalNAc ₃ -7 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-7a A_(d) 2322 ^(m)C_(ds)A_(ds)T_(as)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-10a A_(d) 2322 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) A _(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-13a A_(d) 2322 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) The legend for Table 95 can be found in Example 74. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 96 Antisense inhibition of human TTR in vivo Dosage TTR TTR GalNAc Isis No. (mg/kg) mRNA (% PBS) protein (% BL) cluster CM PBS n/a 100 124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73 GalNAc₃-3a PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a A_(d) 2 33 57 6 17 22 682887 0.6 60 97 GalNAc₃-7a A_(d) 2 36 49 6 12 19 682888 0.6 65 92 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13a A_(d) 2 38 45 6 16 18

Example 91 Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 97 below were tested in a non-terminal, dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 97 or with PBS. Each treatment group consisted of 4 males and 1 female. Prior to the first dose and at various time points thereafter, blood draws were performed to determine plasma Factor VII protein levels. Factor VII protein levels were measured by ELISA. The results presented in Table 98 are the average values for each treatment group relative to the average value for the PBS group at baseline (BL), the measurements taken just prior to the first dose. As illustrated in Table 98, treatment with antisense oligonucleotides lowered Factor VII expression levels in a dose-dependent manner, and the oligonucleotide comprising the GalNAc conjugate was significantly more potent in monkeys compared to the oligonucleotide lacking a GalNAc conjugate.

TABLE 97 Oligonucleotides targeting Factor VII Sequence GalNAc SEQ Isis No. 5′ to 3′ Linkages cluster CM ID No. 407935 A_(es)T_(es)G_(es) ^(m) PS n/a n/a 2323 C_(es)A_(es)T_(ds)G_(ds) G_(ds)T_(ds)G_(ds)A_(ds) T_(ds)G_(ds) ^(m)C_(ds) T_(dsT) _(es) ^(m)C_(es) T_(es)G_(es)A_(e) 686892 GalNAc ₃ - PS GalNAc₃-10a PO 2323 10 _(a-o′)A_(es)T_(es) G_(es) ^(m)C_(es)A_(es) T_(ds)G_(ds)G_(ds) T_(ds)G_(ds)A_(ds) T_(ds)G_(ds) ^(m)C_(ds) T_(ds)T_(es) ^(m)C_(es) T_(es)G_(es)A_(e) The legend for Table 97 can be found in Example 74. The structure of GalNAc₃-10_(a) was shown in Example 46.

TABLE 98 Factor VII plasma protein levels ISIS No. Day Dose (mg/kg) Factor VII (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 46 43 n/a 43 686892 0  3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a 11

Example 92 Antisense Inhibition in Primary Hepatocytes by Antisense Oligonucleotides Targeting Apo-CIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 99, targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or 333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24 hours, the cells were lysed and total RNA was purified using RNeasy (Qiagen). ApoC-III mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) according to standard protocols. IC₅₀ values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide lacking a conjugate.

TABLE 99 Inhibition of mouse APOC-III expression in mouse primary hepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) n/a 13.20 2324 661180 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(d) 1.40 2325 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 0.70 2324 A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 1.70 2324 A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 2.00 2324 A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) PO 1.50 2324 A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(eo)G_(eo) ^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(eo) PO <0.46 2324 A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3 _(a)-_(o′) A _(do) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) A_(d) 1.10 2324 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(d) 2.50 2325 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9, GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown in Example 48, GalNAc₃-l0_(a) was shown in Example 46, GalNAc₃-13_(a) was shown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93 Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 100 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequence (5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 2326 699806 GalNAc ₃ -3 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3a PO 2326 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699807 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 2326 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699809 GalNAc ₃ -7 _(a) - _(o′) T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 2326 T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 699811 GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 2326 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699813 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ds) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 2326 T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 699815 GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 2326 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e) The structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48. Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside (cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO). Supersript “m” indicates 5-methylcytosines.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 100 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-1 mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 101, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the gapmer oligonucleotides comprising a GalNAc conjugate and having wings that are either full cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were also measured, and the average values for each treatment group are shown in Table 101. Body weight is shown as the average percent body weight relative to the baseline body weight (% BL) measured just prior to the oligonucleotide dose.

TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights SRB-1 ISIS Dosage mRNA ALT AST Body weight No. (mg/kg) (% PBS) (U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 18 48 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1 114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104 699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 69 0.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 1 33 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.13 25 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 22 61 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 105 0.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94 Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 102 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequence (5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 2304 T_(es)T_(e) 700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms) n/a n/a 2327 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-3a PO 2304 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 700991 GalNAc ₃ -7 _(a) - _(o′)G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃-7a PO 2327 A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m) Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example 74 for complete table legend. The structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 103 below and show that both the 2′-MOE and 2′-OMe modified oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides lacking a conjugate. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 103 SRB-1 mRNA ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 45 10 17 700991 1 118 3 63 10 14

Example 95 Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 104 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a n/a 2298 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 2298 699782 GalNAc ₃ -7 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-7_(a) PO 2298 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls) ^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 2298 653621 T_(ls) ^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′) -GalNAc ₃ -1a GalNAc₃-1_(a) A_(d) 2299 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/a n/a 2298 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 2298 Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicates a locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74 table legend for other abbreviations. The structure of GalNAc₃-1_(a) was shown previously in Example 9, the structure of GalNAc₃-3_(a) was shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 105 below and show that oligonucleotides comprising a GalNAc conjugate and various bicyclic nucleoside modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising bicyclic nucleoside modifications. Furthermore, the oligonucleotide comprising a GalNAc conjugate and fluoro-HNA modifications was significantly more potent than the parent lacking a conjugate and comprising fluoro-HNA modifications. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 440762 1 104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15 699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 77 10 37 699789 0.1 82 0.3 69 1 26

Example 96 Plasma Protein Binding of Antisense Oligonucleotides Comprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.

TABLE 106 Modified oligonucleotides targeting Apo(a) ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 2321 T_(es) ^(m)C_(e) 693401 T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 2321 T_(es) ^(m)C_(e) 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) GalNAc₃-7_(a) PO 2321 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) GalNAc₃-7_(a) PO 2321 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) See the Example 74 for table legend. The structure of GalNAc₃-7a was shown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regenerated cellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with 300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, then with 3004 of a 300 μg/mL solution of a control oligonucleotide in H₂O and centrifuged at 2000 g for 16 minutes. In order to assess non-specific binding to the filters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mL solution of oligonucleotide in H₂O at pH 7.4 was placed in the pre-conditioned filters and centrifuged at 2000 g for 16 minutes. The unfiltered and filtered samples were analyzed by an ELISA assay to determine the oligonucleotide concentrations. Three replicates were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample is used to determine the percent of oligonucleotide that is recovered through the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-free human volunteers, cynomolgus monkeys, and CD-1 mice, were purchased from Bioreclamation LLC (Westbury, N.Y.). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37° C. for 30 minutes, immediately followed by centrifugation at 2000 g for 16 minutes. Aliquots of filtered and unfiltered spiked plasma samples were analyzed by an ELISA to determine the oligonucleotide concentration in each sample. Three replicates per concentration were used to determine the average percentage of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample is used to determine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (% unbound). The final unbound oligonucleotide values are corrected for non-specific binding by dividing the % unbound by the % recovery for each oligonucleotide. The final % bound oligonucleotide values are determined by subtracting the final % unbound values from 100. The results are shown in Table 107 for the two concentrations of oligonucleotide tested (5 and 150 μg/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a significant impact on plasma protein binding. Furthermore, oligonucleotides with full PS internucleoside linkages and mixed PO/PS linkages both bind plasma proteins, and those with full PS linkages bind plasma proteins to a somewhat greater extent than those with mixed PO/PS linkages.

TABLE 107 Percent of modified oligonucleotide bound to plasma proteins Human plasma Monkey plasma Mouse plasma ISIS 5 150 5 150 5 150 No. μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2 663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.6 89.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.0 94.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.6 93.7 95.6 92.7

Example 97 Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃ Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were designed to target TTR.

TABLE 108 Modified oligonucleotides targeting TTR GalNAc₃ SEQ ID ISIS No. Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m) C_(ds) GalNAc₃-3 A_(d) 2322 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666942 T_(es) ^(m)C_(eo) T_(eo) T_(eo) G_(eo) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-1 A_(d) 2318 A_(eo) T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -3 _(a) 682876 GalNAc ₃ -3 _(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-3 PO 2317 G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682877 GalNAc ₃ -7 _(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-7 PO 2317 G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682878 GalNAc ₃ -10 _(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-10 PO 2317 T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682879 GalNAc ₃ -13 _(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-13 PO 2317 T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682880 GalNAc ₃ -7 _(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7 A_(d) 2322 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682881 GalNAc ₃ -10 _(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-10 A_(d) 2322 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682882 GalNAc ₃ -13 _(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-13 A_(d) 2322 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684056 T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-19 A_(d) 2318 A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The legend for Table 108 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example 48. The structure of GalNAc₃-10_(a) was shown in Example 46. The structure of GalNAc₃-13_(a) was shown in Example 62. The structure of GalNAc₃-19_(a) was shown in Example 70.

Example 98 Evaluation of Pro-Inflammatory Effects of Oligonucleotides Comprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested for pro-inflammatory effects in an hPMBC assay as described in Examples 23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the oligonucleotides.) ISIS 353512 is a high responder used as a positive control, and the other oligonucleotides are described in Tables 83, 95, and 108. The results shown in Table 109 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS internucleoside linkages produced significantly lower pro-inflammatory responses compared to the same oligonucleotides having full PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.

TABLE 109 ISIS No. E_(max)/EC₅₀ GalNAc₃ cluster Linkages CM 353512 3630 n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 682888 0.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99 Binding Affinities of Oligonucleotides Comprising a GalNAc Conjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 110 (see Table 76 for descriptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competitor ligand, α1-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 37° C., and >90% desialylation was confirmed by either sialic acid assay or size exclusion chromatography (SEC). Iodine monochloride was used to iodinate the AGP according to the procedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1):173-81.) In this method, desialylated α1-acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na¹²⁵I, and 1 M glycine in 0.25 M NaOH. After incubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP was separated from free ¹²⁵I by concentrating the mixture twice utilizing a 3 KDMWCO spin column. The protein was tested for labeling efficiency and purity on a HPLC system equipped with an Agilent SEC-3 column (7.8×300mm) and a B-RAM counter. Competition experiments utilizing ¹²⁵I-labeled de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (10⁶ cells/ml) were plated on 6-well plates in 2 ml of appropriate growth media. MEM media supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used. Cells were incubated 16-20 hours @37° C. with 5% and 10% CO₂ respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37° C. with 1 ml competition mix containing appropriate growth media with 2% FBS, 10⁻⁸ M ¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10⁻¹¹ to 10⁻⁵ M. Non-specific binding was determined in the presence of 10⁻² M GalNAc sugar. Cells were washed twice with media without FBS to remove unbound ¹²⁵I-labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen's RLT buffer containing 1% B-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a γ-counter. Non-specific binding was subtracted before dividing ¹²⁵I protein counts by the value of the lowest GalNAc-ASO concentration counts. The inhibition curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to calculate the binding affinities (K_(D)'s).

The results in Table 110 were obtained from experiments performed on five different days. Results for oligonucleotides marked with superscript “a” are the average of experiments run on two different days. The results show that the oligonucleotides comprising a GalNAc conjugate group on the 5′-end bound the asialoglycoprotein receptor on human HepG2 cells with 1.5 to 16-fold greater affinity than the oligonucleotides comprising a GalNAc conjugate group on the 3′-end.

TABLE 110 Asialoglycoprotein receptor binding assay results Oligonucleotide end to GalNAc which GalNAc conjugate ISIS No. conjugate is attached K_(D) (nM) 661161^(a) GalNAc₃-3 5′ 3.7 666881^(a) GalNAc₃-10 5′ 7.6 666981 GalNAc₃-7 5′ 6.0 670061 GalNAc₃-13 5′ 7.4 655861^(a) GalNAc₃-1 3′ 11.6 677841^(a) GalNAc₃-19 3′ 60.8

Example 100 Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.

TABLE 111a Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es) GalNAc₃-7a PO 2321 T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo) GalNAc₃-7a PO 2321 T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds) G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo) T_(es)T_(es) ^(m)C_(e) The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Female transgenic mice that express human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an oligonucleotide and dosage listed in Table 111b or with PBS. Each treatment group consisted of 3 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 72 hours, 1 week, and 2 weeks following the first dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the first dose. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 111b are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the oligonucleotides comprising a GalNAc conjugate group exhibited potent reduction in Apo(a) expression. This potent effect was observed for the oligonucleotide that comprises full PS internucleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.

TABLE 111b Apo(a) plasma protein levels Apo(a) at Apo(a) Apo(a) Dosage 72 hours at 1 week at 3 weeks ISIS No. (mg/kg) (% BL) (% BL) (% BL) PBS n/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 23 15 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101 Antisense Inhibition by Oligonucleotides Comprising a GalNAc Cluster Linked via a Stable Moiety

The oligonucleotides listed in Table 112 were tested for inhibition of mouse APOC-III expression in vivo. C57Bl/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 112 or with PBS. Each treatment group consisted of 4 animals. Each mouse treated with ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mouse treated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847 is linked via a stable moiety, a phosphorothioate linkage instead of a readily cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using real-time PCR. APOC-III mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented in Table 112 as the average percent of APOC-III mRNA levels for each treatment group relative to the saline control group. The results show that the oligonucleotides comprising a GalNAc conjugate group were significantly more potent than the oligonucleotide lacking a conjugate group. Furthermore, the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a cleavable moiety (ISIS 680772) was even more potent than the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 112 Modified oligonucleotides targeting mouse APOC-III APOC-III SEQ ISIS Dosage mRNA ID No. Sequences (5′ to 3′) CM (mg/kg) (% PBS No. 440670 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 2324 G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 20 59 60 37 680772 GalNAc ₃-7 _(a-o′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds )T_(ds)T_(ds)A_(ds)G_(ds) PO 0.6 79 2324 G_(ds)G_(ds)A_(ds) ^(m)C_(es )A_(es)G_(es) ^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃-7 _(a-s′) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds) n/a 0.6 83 2324 G_(ds)G_(ds)A_(ds) ^(m)C_(es )A_(es)G_(es) ^(m)C_(es)A_(e) (PS) 2 73 6 40 20 28

The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102 Distribution in Liver of Antisense Oligonucleotides Comprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 36) that does not comprise a GalNAc conjugate and ISIS 655861 (see Table 36) that does comprise a GalNAc conjugate was evaluated. Male Balb/c mice were subcutaneously injected once with ISIS 353382 or 655861 at a dosage listed in Table 113. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655861, which consisted of 2 animals. The animals were sacrificed 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide molecules per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide probe used to detect the antisense oligonucleotides. The results presented in Table 113 are the average concentrations of oligonucleotide for each treatment group in units of millions of oligonucleotide molecules per cell. The results show that at equivalent doses, the oligonucleotide comprising a GalNAc conjugate was present at higher concentrations in the total liver and in hepatocytes than the oligonucleotide that does not comprise a GalNAc conjugate. Furthermore, the oligonucleotide comprising a GalNAc conjugate was present at lower concentrations in non-parenchymal liver cells than the oligonucleotide that does not comprise a GalNAc conjugate. And while the concentrations of ISIS 655861 in hepatocytes and non-parenchymal liver cells were similar per cell, the liver is approximately 80% hepatocytes by volume. Thus, the majority of the ISIS 655861 oligonucleotide that was present in the liver was found in hepatocytes, whereas the majority of the ISIS 353382 oligonucleotide that was present in the liver was found in non-parenchymal liver cells.

TABLE 113 Concentration in whole Concentration in Concentration in non- ISIS Dosage liver (molecules*10{circumflex over ( )}6 hepatocytes parenchymal liver cells No. (mg/kg) per cell) (molecules*10{circumflex over ( )}6 per cell) (molecules*10{circumflex over ( )}6 per cell) 353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1

Example 103 Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 114 below were tested in a single dose study for duration of action in mice.

TABLE 114 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 2296 T_(es)A_(es)T_(e) 663084 GalNAc ₃-3 _(a)-_(o′) A _(do)A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 2312 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo )T_(es)A_(es)T_(e) 679241 A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 2297 T_(es)A_(es)T_(eo)A_(do′)-GalNAc ₃-19 _(a) The structure of GalNAc₃-3_(a) was shown in Example 39, and GalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 114 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 115 are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels. A comparison of the results in Table 71 of example 79 with the results in Table 115 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate internucleoside linkages exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.

TABLE 115 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point Triglyc- APOC-III (days erides protein ISIS Dosage post- (% base- (% base- GalNAc₃ No. (mg/kg) dose) line) line) Cluster CM PBS n/a 3 96 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 72 88 304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 42 82 92 663084 10 3 35 28 GalNAc₃-3a A_(d) 7 23 24 14 23 26 21 23 29 28 30 22 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃-19a A_(d) 7 31 28 14 30 22 21 36 34 28 48 34 35 50 45 42 72 64

Example 104 Synthesis of Oligonucleotides Comprising a 5′-GalNAc₂ Conjugate

Compound 120 is commercially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF (10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction. After 3 h, the reaction mixture was poured into 100 mL of 1 M NaHSO₄ and extracted with 2×50 mL ethyl acetate. Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and 2× brine, dried with Na₂SO₄, filtered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) to yield compound 231. LCMS and NMR were consistent with the structure. Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 mL) and trifluoracetic acid (10 mL) was added. After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3×10 mL). The residue was dried under reduced pressure to yield compound 232 as the trifuloracetate salt. The synthesis of compound 166 is described in Example 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and N,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirred at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was extracted with EtOAc (2×100 mL). The organic phase was separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 M NaHSO₄ (3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield compound 233. LCMS and NMR were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt % Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The reaction mixture was filtered through a pad of Celite and concentrated to yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolved in DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring at room temperature the reaction mixture was poured into water (40 mL) and extracted with EtOAc (2×50 mL). A standard work-up was completed as described above to yield compound 234. LCMS and NMR were consistent with the structure. Oligonucleotide 235 was prepared using the general procedure described in Example 46. The GalNAc₂ cluster portion (GalNAc₂-24_(a)) of the conjugate group GalNAc₂-24 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₂-24 (GalNAc₂-24_(a)-CM) is shown below:

Example 105 Synthesis of Oligonucleotides Comprising a GalNAc₁-25 Conjugate

The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared using the general procedure described in Example 46. Alternatively, oligonucleotide 236 was synthesized using the scheme shown below, and compound 238 was used to form the oligonucleotide 236 using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate group GalNAc₁-25 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106 Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₂ or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested in dose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222, or 708561; or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the ED₅₀ results are presented in Tables 116 and 117. Although previous studies showed that trivalent GalNAc-conjugated oligonucleotides were significantly more potent than divalent GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than monovalent GalNAc conjugated oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides comprising monovalent, divalent, and trivalent GalNAc clusters lowered SRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No. Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 2298 686221 GalNAc ₂-24 _(a)-_(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 2302 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃-13 _(a)-_(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 2302 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) See Example 93 for table legend. The structure of GalNAc₃-13a was shown in Example 62, and the structure of GalNAc₂-24a was shown in Example 104.

TABLE 117 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No. Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 2298 708561 GalNAc ₁-25 _(a)-_(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 2298 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) See Example 93 for table legend. The structure of GalNAc₁-25a was shown in Example 105.

The concentrations of the oligonucleotides in Tables 116 and 117 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 117a and 117b below are the average total antisense oligonucleotide tissues levels for each treatment group, as measured by UV in units of μg oligonucleotide per gram of liver tissue. The results show that the oligonucleotides comprising a GalNAc conjugate group accumulated in the liver at significantly higher levels than the same dose of the oligonucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc ligands in their respective conjugate groups all accumulated in the liver at similar levels. This result is surprising in view of the Khorev et al. literature reference cited above and is consistent with the activity data shown in Tables 116 and 117 above.

TABLE 117a Liver concentrations of oligonucleotides comprising a GalNAc₂ or GalNAc₃ conjugate group Dosage Antisense ISIS No. (mg/kg) oligonucleotide (μg/g) GalNAc cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1 686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5 GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 117b Liver concentrations of oligonucleotides comprising a GalNAc₁ conjugate group Dosage Antisense GalNAc ISIS No. (mg/kg) oligonucleotide (μg/g) cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7 708561 0.2 0.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107 Synthesis of Oligonucleotides Comprising a GalNAc₁-26 or GalNAc₁-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. The resulting amide containing compound is phosphitylated, then added to the 5′-end of an oligonucleotide using procedures described in Example 10. The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate group GalNAc₁-26 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of an oligonucleotide, the amide formed from the reaction of compounds 47 and 64 is added to a solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate group GalNAc₁-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108 Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a single dose study in mice.

TABLE 118 Modified ASOs targeting APO(a) ISIS SEQ No. Sequences (5′ to 3′) GalNAc₃ Cluster CM ID No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) n/a n/a 2321 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251 GalNAc ₃-7 _(a)-_(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 2321 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃-3 _(a)-_(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 2321 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃-10 _(a)-_(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 2321 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃-7 _(a)-_(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 2321 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃-13 _(a)-_(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 2321 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681260 T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 2328 T_(es)T_(es) ^(m)C_(eo)A_(do′)-GalNAc ₃-19 The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 119 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 1 week following the first dose. Additional blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 119 are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides reduced Apo(a) protein expression. Furthermore, the oligonucleotides comprising a GalNAc conjugate group exhibited even more potent reduction in Apo(a) expression than the oligonucleotide that does not comprise a conjugate group.

TABLE 119 Apo(a) plasma protein levels Apo(a) at 1 week ISIS No. Dosage (mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 681256 10 17 681257 10 24 681258 10 22 681260 10 26

Example 109 Synthesis of Oligonucleotides Comprising a GalNAc₁-28 or GalNAc₁-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures described in Example 10 to synthesize the oligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of the conjugate group GalNAc₁-28 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-28 (GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of an oligonucleotide, procedures similar to those described in Example 71 are used to form the hydroxyl intermediate, which is then added to the solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate group GalNAc₁-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110 Synthesis of Oligonucleotides Comprising a GalNAc₁-30 Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of the conjugate group GalNAc₁-30 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, Y is part of the cleavable moiety. In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₁-30_(a) is shown below:

Example 111 Synthesis of Oligonucleotides Comprising a GalNAc₂-31 or GalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂ cluster portion (GalNAc₂-31_(a)) of the conjugate group GalNAc₂-31 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₂-31_(a) is shown below:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate is shown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of the conjugate group GalNAc₂-32 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5′-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₂-32_(a) is shown below:

Example 112 Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized with a GalNAc₁ conjugate group in order to further test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

TABLE 120 GalNAc SEQ ISIS No. Sequence (5′ to 3′) cluster CM ID NO. 711461 GalNAc ₁-25 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-25_(a) A_(d) 2306 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711462 GalNAc ₁-25 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds) GalNAc₁-25_(a) PO 2304 A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711463 GalNAc ₁-25 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-25_(a) PO 2304 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 711465 GalNAc ₁-26 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-26_(a) A_(d) 2306 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711466 GalNAc ₁-26 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds) GalNAc₁-26_(a) PO 2304 A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711467 GalNAc ₁-26 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-26_(a) PO 2304 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 711468 GalNAc ₁-28 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-28_(a) A_(d) 2306 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711469 GalNAc ₁-28 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds) GalNAc₁-28_(a) PO 2304 A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711470 GalNAc ₁-28 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-28_(a) PO 2304 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 713844 G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-27_(a) PO 2304 T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁-27 _(a) 713845 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-27_(a) PO 2304 T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁-27 _(a) 713846 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-27_(a) A_(d) 2305 T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁-27 _(a) 713847 G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-29_(a) PO 2304 T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁-29 _(a) 713848 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-29_(a) PO 2304 T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁-29 _(a) 713849 G_(es ) ^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-29_(a) A_(d) 2305 T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁-29 _(a) 713850 G_(es ) ^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-29_(a) A_(d) 2305 T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁-29 _(a)

Example 113 Antisense Oligonucleotides Targeting Growth Hormone Receptor and Comprising a GalNAc Cluster

The oligonucleotides in Table 121 were designed to target human growth hormone receptor (GHR).

TABLE 121 Sequences (5′ to 3′) SEQ ID No. GalNAc ₃-3-^(m)C_(es) ^(m)C_(es)A_(es) ^(m)C_(es) ^(m)C_(es)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-3-^(m)C_(es) ^(c)C_(eo)A_(eo) ^(m)C_(eo) ^(m)C_(eo)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(eo)A_(eo)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-7-^(m)C_(es) ^(m)C_(es)A_(es) ^(m)C_(es) ^(m)C_(es)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-7-^(m)C_(es) ^(m)C_(eo)A_(eo) ^(m)C_(eo) ^(m)C_(eo)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(eo)A_(eo)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-10-^(m)C_(es) ^(m)C_(es)A_(es) ^(m)C_(es) ^(m)C_(es)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-10-^(m)C_(es) ^(m)C_(eo)A_(eo) ^(m)C_(eo) ^(m)C_(eo)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(eo)A_(eo)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-13-^(m)C_(es) ^(m)C_(es)A_(es) ^(m)C_(es) ^(m)C_(es)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 703 GalNAc ₃-13-^(m)C_(es) ^(m)C_(eo)A_(eo) ^(m)C_(eo) ^(m)C_(eo)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(eo)A_(eo)G_(es) ^(m)C_(es)A_(e) 703 ^(m)C_(es) ^(m)C_(es)A_(es) ^(m)C_(es) ^(m)C_(es)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(es)A_(es)G_(es) ^(m)C_(es)A_(e)-GalNAc ₃-19 703 ^(m)C_(es) ^(m)C_(eo)A_(eo) ^(m)C_(eo) ^(m)C_(eo)T_(ds)T_(ds)T_(ds)G_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)A_(ds)T_(eo)A_(eo)G_(es) ^(m)C_(es)A_(e)-GalNAc ₃-19 703

Example 114 Antisense Inhibition of Human Growth Hormone Receptor in Hep3B Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting a growth hormone receptor (GHR) nucleic acid and were tested for their effects on GHR mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB (forward sequence CGAGTTCAGTGAGGTGCTCTATGT, designated herein as SEQ ID NO: 2329; reverse sequence AAGAGCCATGGAAAGTAGAAATCTTC, designated herein as SEQ ID NO: 2330; probe sequence TTCCTCAGATGAGCCAATT, designated herein as SEQ ID NO: 2331) was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE or 3-10-4 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and four nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human GHR mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000163.4) or the human GHR genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene.

TABLE 122 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Start Stop Target % Start Stop ID ISIS NO Site Site Region Sequence inhibition Site Site NO 523266 164 183 Exon 1 ACCTCCGAGCTTCGCCTCTG 64   3040   3059 20 523267 171 190 Exon- CTGTAGGACCTCCGAGCTTC 31 n/a n/a 21 exon junction 523268 178 197 Exon- TCCATACCTGTAGGACCTCC 37 n/a n/a 22 exon junction 523271 206 225 Exon 2 TGCCAAGGTCAACAGCAGCT 80 144990 145009 23 523272 213 232 Exon 2 CTGCCAGTGCCAAGGTCAAC 53 144997 145016 24 523273 220 239 Exon 2 CTTGATCCTGCCAGTGCCAA 49 145004 145023 25 523274 227 246 Exon 2 AGCATCACTTGATCCTGCCA 67 145011 145030 26 523275 234 253 Exon 2 CAGAAAAAGCATCACTTGAT 0 145018 145037 27 523276 241 260 Exon 2 TCACTTCCAGAAAAAGCATC 1 145025 145044 28 523284 361 380 Exon 4 GTCTCTCGCTCAGGTGAACG 48 268024 268043 29 523285 368 387 Exon 4 TGAAAAAGTCTCTCGCTCAG 15 268031 268050 30 523286 375 394 Exon 4 AGTGGCATGAAAAAGTCTCT 14 268038 268057 31 523287 382 401 Exon 4 TCTGTCCAGTGGCATGAAAA 4 268045 268064 32 523301 625 644 Exon 6 GGATCTGGTTGCACTATTTC 36 n/a n/a 33 523302 632 651 Exon 6 AATGGGTGGATCTGGTTGCA 28 278926 278945 34 523303 647 666 Exon 6 AGTCCAGTTGAGGGCAATGG 26 278941 278960 35 523304 654 673 Exon 6 TCAGTAAAGTCCAGTTGAGG 0 278948 278967 36 523305 675 694 Exon 6 GAATCCCAGTTAAACTGACG 19 278969 278988 37 523306 682 701 Exon 6 TCTGCATGAATCCCAGTTAA 39 278976 278995 38 523309 736 755 Exon 6 ATCCATCCTTTCTGAATATC 34 279030 279049 39 523310 743 762 Exon 6 CAGAACCATCCATCCTTTCT 31 279037 279056 40 523311 750 769 Exon 6 CATACTCCAGAACCATCCAT 44 279044 279063 41 523312 757 776 Exon 6 TGAAGTTCATACTCCAGAAC 23 279051 279070 42 523313 764 783 Exon 6 TTTGTATTGAAGTTCATACT 6 279058 279077 43 523314 771 790 Exon 6 TTACTTCTTTGTATTGAAGT 0 279065 279084 44 523315 778 797 Exon 6 GTTTCATTTACTTCTTTGTA 3 279072 279091 45 523316 785 804 Exon 6 CCATTTAGTTTCATTTACTT 0 279079 279098 46 523317 792 811 Exon 4- TCATTTTCCATTTAGTTTCA 19 n/a n/a 47 exon 5 junction 523323 862 881 Exon 7 ACACGCACTTCATATTCCTT 63 290360 290379 48 523324 869 888 Exon 7 GGATCTCACACGCACTTCAT 80 290367 290386 49 523328 926 945 Exon 7 AAGTGTTACATAGAGCACCT 56 290424 290443 50 523329 933 952 Exon 7 TCTGAGGAAGTGTTACATAG 53 290431 290450 51 523330 957 976 Exon 7 CTTCTTCACATGTAAATTGG 32 290455 290474 52 523331 964 983 Exon 5- TAGAAATCTTCTTCACATGT 4 n/a n/a 53 exon 6 junction 523332 971 990 Exon 5- TGGAAAGTAGAAATCTTCTT 9 n/a n/a 54 exon 6 junction 523333 978 997 Exon 8 AGAGCCATGGAAAGTAGAAA 46 292532 292551 55 523334 985 1004 Exon 8 ATAATTAAGAGCCATGGAAA 0 292539 292558 56

TABLE 123 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting exonic regions of SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop Target % Start Stop ID NO Site Site Region Sequence inhibition Site Site NO 523421 2072 2091 exon 10 CAGTTGGTCTGTGCTCACAT 76 298489 298508 57 533002 207 226 exon 2 GTGCCAAGGTCAACAGCAGC 63 144991 145010 58 533003 208 227 exon 2 AGTGCCAAGGTCAACAGCAG 62 144992 145011 59 533004 225 244 exon 2 CATCACTTGATCCTGCCAGT 53 145009 145028 60 533005 226 245 exon 2 GCATCACTTGATCCTGCCAG 80 145010 145029 61 533006 228 247 exon 2 AAGCATCACTTGATCCTGCC 75 145012 145031 62 533007 229 248 exon 2 AAAGCATCACTTGATCCTGC 61 145013 145032 63 533019 867 886 exon 7 ATCTCACACGCACTTCATAT 35 290365 290384 64 533020 868 887 exon 7 GATCTCACACGCACTTCATA 47 290366 290385 65 533021 870 889 exon 7 TGGATCTCACACGCACTTCA 86 290368 290387 66 533022 871 890 exon 7 TTGGATCTCACACGCACTTC 70 290369 290388 67 533037 1360 1379 exon 10 TCCAGAATGTCAGGTTCACA 59 297777 297796 68 533038 1361 1380 exon 10 CTCCAGAATGTCAGGTTCAC 74 297778 297797 69 533039 1363 1382 exon 10 GTCTCCAGAATGTCAGGTTC 45 297780 297799 70 533040 1364 1383 exon 10 AGTCTCCAGAATGTCAGGTT 51 297781 297800 71 533042 1525 1544 exon 10 GCTTGGATAACACTGGGCTG 41 297942 297961 72 533043 1526 1545 exon 10 TGCTTGGATAACACTGGGCT 46 297943 297962 73 533044 1528 1547 exon 10 TCTGCTTGGATAACACTGGG 55 297945 297964 74 533045 1529 1548 exon 10 CTCTGCTTGGATAACACTGG 47 297946 297965 75 533046 1530 1549 exon 10 TCTCTGCTTGGATAACACTG 54 297947 297966 76 533047 1744 1763 exon 10 CAGAGTGAGACCATTTCCGG 47 298161 298180 77 533048 1745 1764 exon 10 GCAGAGTGAGACCATTTCCG 60 298162 298181 78 533049 1747 1766 exon 10 TGGCAGAGTGAGACCATTTC 65 298164 298183 79 533050 1748 1767 exon 10 TTGGCAGAGTGAGACCATTT 47 298165 298184 80 533051 1749 1768 exon 10 CTTGGCAGAGTGAGACCATT 30 298166 298185 81 533066 2685 2704 exon 10 CAGTGTGTAGTGTAATATAA 53 299102 299121 82 533067 2686 2705 exon 10 ACAGTGTGTAGTGTAATATA 68 299103 299122 83 533068 2688 2707 exon 10 ACACAGTGTGTAGTGTAATA 62 299105 299124 84 533069 2689 2708 exon 10 TACACAGTGTGTAGTGTAAT 55 299106 299125 85 533070 2690 2709 exon 10 GTACACAGTGTGTAGTGTAA 50 299107 299126 86 533071 3205 3224 exon 10 TGTACCTTATTCCCTTCCTG 68 299622 299641 87 533072 3206 3225 exon 10 TTGTACCTTATTCCCTTCCT 61 299623 299642 88 533073 3208 3227 exon 10 TCTTGTACCTTATTCCCTTC 60 299625 299644 89 533074 3209 3228 exon 10 TTCTTGTACCTTATTCCCTT 46 299626 299645 90

TABLE 124 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 ISIS Start Stop Target % Start Stop SEQ NO Site Site Region Sequence inhibition Site Site ID NO 532174 n/a n/a Intron 1 ACATGTACCCAAACCAACAC 37 18731 18750 91 533086 3210 3229 Exon 10 CTTCTTGTACCTTATTCCCT 72 299627 299646 92 533087 3212 3231 Exon 10 TGCTTCTTGTACCTTATTCC 77 299629 299648 93 533088 3213 3232 Exon 10 ATGCTTCTTGTACCTTATTC 63 299630 299649 94 533089 3215 3234 Exon 10 AAATGCTTCTTGTACCTTAT 67 299632 299651 95 533090 3216 3235 Exon 10 AAAATGCTTCTTGTACCTTA 50 299633 299652 96 533091 3217 3236 Exon 10 CAAAATGCTTCTTGTACCTT 44 299634 299653 97 533092 3518 3537 Exon 10 CTTCTGAATGCTTGCTTTGA 29 299935 299954 98 533093 3519 3538 Exon 10 TCTTCTGAATGCTTGCTTTG 47 299936 299955 99 533094 3521 3540 Exon 10 TTTCTTCTGAATGCTTGCTT 63 299938 299957 100 533095 3522 3541 Exon 10 TTTTCTTCTGAATGCTTGCT 51 299939 299958 101 533096 3523 3542 Exon 10 TTTTTCTTCTGAATGCTTGC 34 299940 299959 102 533097 4041 4060 Exon 10 TGCGATAAATGGGAAATACT 36 300458 300477 103 533098 4042 4061 Exon 10 CTGCGATAAATGGGAAATAC 52 300459 300478 104 533099 4043 4062 Exon 10 TCTGCGATAAATGGGAAATA 41 300460 300479 105 533100 4045 4064 Exon 10 GGTCTGCGATAAATGGGAAA 40 300462 300481 106 533101 4046 4065 Exon 10 AGGTCTGCGATAAATGGGAA 39 300463 300482 107 533102 4048 4067 Exon 10 AAAGGTCTGCGATAAATGGG 34 300465 300484 108 533103 4049 4068 Exon 10 AAAAGGTCTGCGATAAATGG 35 300466 300485 109 533104 4050 4069 Exon 10 AAAAAGGTCTGCGATAAATG 15 300467 300486 110 533115 n/a n/a Intron 1 CATGAAGGCCACTCTTCCAA 63 12777 12796 111 533116 n/a n/a Intron 1 CCATGAAGGCCACTCTTCCA 78 12778 12797 112 533117 n/a n/a Intron 1 CCCATGAAGGCCACTCTTCC 71 12779 12798 113 533118 n/a n/a Intron 1 TGCCCATGAAGGCCACTCTT 66 12781 12800 114 533119 n/a n/a Intron 1 TTGCCCATGAAGGCCACTCT 60 12782 12801 115 533120 n/a n/a Intron 1 GTTGCCCATGAAGGCCACTC 74 12783 12802 116 533121 n/a n/a Intron 1 GGTCTTTCATGAATCAAGCT 79 17927 17946 117 533122 n/a n/a Intron 1 TGGTCTTTCATGAATCAAGC 83 17928 17947 118 533123 n/a n/a Intron 1 ATGGTCTTTCATGAATCAAG 83 17929 17948 119 533124 n/a n/a Intron 1 TGATGGTCTTTCATGAATCA 78 17931 17950 120 533125 n/a n/a Intron 1 CTGATGGTCTTTCATGAATC 82 17932 17951 121 533126 n/a n/a Intron 1 GCTGATGGTCTTTCATGAAT 74 17933 17952 122 533127 n/a n/a Intron 1 GTACCCAAACCAACACTAAT 57 18727 18746 123 533128 n/a n/a Intron 1 TGTACCCAAACCAACACTAA 65 18728 18747 124 533129 n/a n/a Intron 1 ATGTACCCAAACCAACACTA 64 18729 18748 125 533130 n/a n/a Intron 1 GACATGTACCCAAACCAACA 63 18732 18751 126 533131 n/a n/a Intron 1 AGACATGTACCCAAACCAAC 81 18733 18752 127 533132 n/a n/a Intron 1 AGGAATGGAAAACCAAATAT 49 26494 26513 128 533133 n/a n/a Intron 1 CAGGAATGGAAAACCAAATA 74 26495 26514 129 121986 122005 533134 n/a n/a Intron 1 TCAGGAATGGAAAACCAAAT 73 26496 26515 130 121987 122006 533135 n/a n/a Intron 1 ACTCAGGAATGGAAAACCAA 77 26498 26517 131 113032 113051 121989 122008 533136 n/a n/a Intron 1 AACTCAGGAATGGAAAACCA 79 26499 26518 132 113033 113052 121990 122009 533137 n/a n/a Intron 1 TAACTCAGGAATGGAAAACC 67 26500 26519 133 113034 113053 121991 122010 533138 n/a n/a Intron 1 CAAAATTACTGCAGTCACAG 67 39716 39735 134 533139 n/a n/a Intron 1 ACAAAATTACTGCAGTCACA 81 39717 39736 135 533140 n/a n/a Intron 1 TACAAAATTACTGCAGTCAC 81 39718 39737 136 533141 n/a n/a Intron 1 CATACAAAATTACTGCAGTC 67 39720 39739 137 533142 n/a n/a Intron 1 ACATACAAAATTACTGCAGT 48 39721 39740 138 533143 n/a n/a Intron 1 AACATACAAAATTACTGCAG 53 39722 39741 139 533144 n/a n/a Intron 1 TTTTAGTATGAACCTTAAAA 0 42139 42158 140 533145 n/a n/a Intron 1 CTTTTAGTATGAACCTTAAA 38 42140 42159 141 533146 n/a n/a Intron 1 TCTTTTAGTATGAACCTTAA 57 42141 42160 142 533147 n/a n/a Intron 1 AATCTTTTAGTATGAACCTT 60 42143 42162 143 533148 n/a n/a Intron 1 CAATCTTTTAGTATGAACCT 70 42144 42163 144 533149 n/a n/a Intron 1 ACAATCTTTTAGTATGAACC 60 42145 42164 145 533150 n/a n/a Intron 1 AAGTTATGTGACTCTGAGCA 67 43174 43193 146 533151 n/a n/a Intron 1 CAAGTTATGTGACTCTGAGC 67 43175 43194 147 533152 n/a n/a Intron 1 TCAAGTTATGTGACTCTGAG 63 43176 43195 148 533153 n/a n/a Intron 1 AGTTCTCCATTAGGGTTCTG 83 50948 50967 149 533154 n/a n/a Intron 1 TAGTTCTCCATTAGGGTTCT 76 50949 50968 150 533155 n/a n/a Intron 1 ATAGTTCTCCATTAGGGTTC 51 50950 50969 151 533156 n/a n/a Intron 1 AAGCAGGTTGGCAGACAGAC 79 53467 53486 152 533157 n/a n/a Intron 1 GAAGCAGGTTGGCAGACAGA 60 53468 53487 153 533158 n/a n/a Intron 1 GGAAGCAGGTTGGCAGACAG 67 53469 53488 154 533159 n/a n/a Intron 1 TCTTCTTGTGAGCTGGCTTC 61 64882 64901 155 533160 n/a n/a Intron 1 GTCTTCTTGTGAGCTGGCTT 83 64883 64902 156 533161 n/a n/a Intron 1 AGTCTTCTTGTGAGCTGGCT 81 64884 64903 157

TABLE 125 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID ID ID NO: 1 NO: 1 SEQ ID NO: 2 SEQ ISIS Start Stop Target % NO: 2 Stop ID NO Site Site Region Sequence inhibition Start Site Site NO 533133 n/a n/a Intron 1 CAGGAATGGAAAACCAAATA 76 26495 26514 129 121986 122005 533134 n/a n/a Intron 1 TCAGGAATGGAAAACCAAAT 83 26496 26515 130 121987 122006 533174 n/a n/a Intron 1 TAAGTCTTCTTGTGAGCTGG 73 64886 64905 158 533175 n/a n/a Intron 1 TTAAGTCTTCTTGTGAGCTG 58 64887 64906 159 533176 n/a n/a Intron 1 ATTAAGTCTTCTTGTGAGCT 51 64888 64907 160 533177 n/a n/a Intron 1 TCTCTTCCACTCACATCCAT 72 65989 66008 161 533178 n/a n/a Intron 1 GTCTCTTCCACTCACATCCA 86 65990 66009 162 533179 n/a n/a Intron 1 AGTCTCTTCCACTCACATCC 80 65991 66010 163 533180 n/a n/a Intron 1 TAAGTATTTGTAGCAGTTGC 31 78195 78214 164 533181 n/a n/a Intron 1 CTAAGTATTTGTAGCAGTTG 14 78196 78215 165 533182 n/a n/a Intron 1 GCTAAGTATTTGTAGCAGTT 59 78197 78216 166 533183 n/a n/a Intron 1 TGGCTAAGTATTTGTAGCAG 34 78199 78218 167 533184 n/a n/a Intron 1 TTGGCTAAGTATTTGTAGCA 18 78200 78219 168 533185 n/a n/a Intron 1 TTTGGCTAAGTATTTGTAGC 21 78201 78220 169 533186 n/a n/a Intron 1 AAAATGTCAACAGTGCATAG 61 80636 80655 170 533187 n/a n/a Intron 1 CAAAATGTCAACAGTGCATA 78 80637 80656 171 533188 n/a n/a Intron 1 CCAAAATGTCAACAGTGCAT 85 80638 80657 172 533189 n/a n/a Intron 1 GCCCAAAATGTCAACAGTGC 82 80640 80659 173 533190 n/a n/a Intron 1 GGCCCAAAATGTCAACAGTG 60 80641 80660 174 533191 n/a n/a Intron 1 TGGCCCAAAATGTCAACAGT 31 80642 80661 175 533192 n/a n/a Intron 1 CAGAATCTTCTCTTTGGCCA 66 98624 98643 176 533193 n/a n/a Intron 1 GCAGAATCTTCTCTTTGGCC 81 98625 98644 177 533194 n/a n/a Intron 1 TGCAGAATCTTCTCTTTGGC 72 98626 98645 178 533195 n/a n/a Intron 1 TTTGCAGAATCTTCTCTTTG 33 98628 98647 179 533196 n/a n/a Intron 1 ATTTGCAGAATCTTCTCTTT 27 98629 98648 180 533197 n/a n/a Intron 1 AATTTGCAGAATCTTCTCTT 38 98630 98649 181 533198 n/a n/a Intron 1 ATAAAGCTATGCCATAAAGC 37 99478 99497 182 533199 n/a n/a Intron 1 CATAAAGCTATGCCATAAAG 14 99479 99498 183 533200 n/a n/a Intron 1 CCATAAAGCTATGCCATAAA 30 99480 99499 184 533201 n/a n/a Intron 1 GACCATAAAGCTATGCCATA 54 99482 99501 185 533202 n/a n/a Intron 1 TGACCATAAAGCTATGCCAT 64 99483 99502 186 533203 n/a n/a Intron 1 CTGACCATAAAGCTATGCCA 61 99484 99503 187 533204 n/a n/a Intron 1 CAAAAAGTTGAGCTGAGAAA 0 101078 101097 188 533205 n/a n/a Intron 1 CCAAAAAGTTGAGCTGAGAA 28 101079 101098 189 533206 n/a n/a Intron 1 CCCAAAAAGTTGAGCTGAGA 52 101080 101099 190 533207 n/a n/a Intron 1 CACCCAAAAAGTTGAGCTGA 60 101082 101101 191 533208 n/a n/a Intron 1 ACACCCAAAAAGTTGAGCTG 34 101083 101102 192 533209 n/a n/a Intron 1 TACACCCAAAAAGTTGAGCT 36 101084 101103 193 533210 n/a n/a Intron 1 CTTTTAATGGCACCCAAGCA 41 103566 103585 194 533211 n/a n/a Intron 1 GCTTTTAATGGCACCCAAGC 54 103567 103586 195 533212 n/a n/a Intron 1 TGCTTTTAATGGCACCCAAG 67 103568 103587 196 533213 n/a n/a Intron 1 AATGCTTTTAATGGCACCCA 73 103570 103589 197 533214 n/a n/a Intron 1 AAATGCTTTTAATGGCACCC 73 103571 103590 198 533215 n/a n/a Intron 1 GAAATGCTTTTAATGGCACC 41 103572 103591 199 533216 n/a n/a Intron 1 TAATTCTTAAGGGCCCTCTG 36 106963 106982 200 533217 n/a n/a Intron 1 ATAATTCTTAAGGGCCCTCT 45 106964 106983 201 533218 n/a n/a Intron 1 CATAATTCTTAAGGGCCCTC 50 106965 106984 202 533219 n/a n/a Intron 1 AGCATAATTCTTAAGGGCCC 48 106967 106986 203 533220 n/a n/a Intron 1 TAGCATAATTCTTAAGGGCC 52 106968 106987 204 533221 n/a n/a Intron 1 TTAGCATAATTCTTAAGGGC 28 106969 106988 205 533222 n/a n/a Intron 1 AGGAATGGAAAACCAAACAT 13 113028 113047 206 533223 n/a n/a Intron 1 CAGGAATGGAAAACCAAACA 64 113029 113048 207 533224 n/a n/a Intron 1 TCAGGAATGGAAAACCAAAC 61 113030 113049 208 533225 n/a n/a Intron 1 AGGAATGGAAAACCAAATAC 18 121985 122004 209 533226 n/a n/a Intron 1 CATGACTATGTTCTGGCAAG 37 125591 125610 210 533227 n/a n/a Intron 1 ACATGACTATGTTCTGGCAA 44 125592 125611 211 533228 n/a n/a Intron 1 CACATGACTATGTTCTGGCA 63 125593 125612 212 533229 n/a n/a Intron 1 GTCACATGACTATGTTCTGG 47 125595 125614 213 533230 n/a n/a Intron 1 GGTCACATGACTATGTTCTG 49 125596 125615 214 533231 n/a n/a Intron 1 TGGTCACATGACTATGTTCT 30 125597 125616 215 533232 n/a n/a Intron 2 CTGAATTCTGAGCTCTGGAA 73 145428 145447 216 533233 n/a n/a Intron 2 CCTGAATTCTGAGCTCTGGA 88 145429 145448 217 533234 n/a n/a Intron 2 GCCTGAATTCTGAGCTCTGG 92 145430 145449 218 533235 n/a n/a Intron 2 AAGCCTGAATTCTGAGCTCT 83 145432 145451 219 533236 n/a n/a Intron 2 CAAGCCTGAATTCTGAGCTC 68 145433 145452 220 533237 n/a n/a Intron 2 ACAAGCCTGAATTCTGAGCT 81 145434 145453 221 533238 n/a n/a Intron 2 GGATCTCAGCTGCAATTCTT 72 146235 146254 222 533239 n/a n/a Intron 2 AGGATCTCAGCTGCAATTCT 53 146236 146255 223 533240 n/a n/a Intron 2 GAGGATCTCAGCTGCAATTC 69 146237 146256 224 533241 n/a n/a Intron 2 CAGAGGATCTCAGCTGCAAT 69 146239 146258 225 533242 n/a n/a Intron 2 GCAGAGGATCTCAGCTGCAA 76 146240 146259 226 533243 230 249 Exon 2 AAAAGCATCACTTGATCCTG 23 145014 145033 227

TABLE 126 Inhibition of GHR mRNA by 3-10-4 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 ISIS Start Stop Target % Start Stop SEQ NO Site Site Region Sequence inhibition Site Site ID NO 539284 206 222 Exon 2 CAAGGTCAACAGCAGCT 62 144990 145006 228 539285 207 223 Exon 2 CCAAGGTCAACAGCAGC 74 144991 145007 229 539286 208 224 Exon 2 GCCAAGGTCAACAGCAG 73 144992 145008 230 539290 869 885 Exon 7 TCTCACACGCACTTCAT 29 290367 290383 231 539291 870 886 Exon 7 ATCTCACACGCACTTCA 51 290368 290384 232 539292 871 887 Exon 7 GATCTCACACGCACTTC 56 290369 290385 233 539299 n/a n/a Intron 1 CTTTCATGAATCAAGCT 63 17927 17943 234 539300 n/a n/a Intron 1 TCTTTCATGAATCAAGC 49 17928 17944 235 539301 n/a n/a Intron 1 GTCTTTCATGAATCAAG 61 17929 17945 236 539302 n/a n/a Intron 1 GGTCTTTCATGAATCAA 93 17930 17946 237 539303 n/a n/a Intron 1 ATGGTCTTTCATGAATC 74 17932 17948 238 539304 n/a n/a Intron 1 GATGGTCTTTCATGAAT 56 17933 17949 239 539305 n/a n/a Intron 1 TATATCAATATTCTCCC 42 21820 21836 240 539306 n/a n/a Intron 1 TTATATCAATATTCTCC 33 21821 21837 241 539307 n/a n/a Intron 1 GTTATATCAATATTCTC 12 21822 21838 242 539308 n/a n/a Intron 1 TTTCTTTAGCAATAGTT 21 22518 22534 243 539309 n/a n/a Intron 1 CTTTCTTTAGCAATAGT 38 22519 22535 244 539310 n/a n/a Intron 1 GCTTTCTTTAGCAATAG 39 22520 22536 245 539311 n/a n/a Intron 1 AGGAATGGAAAACCAAA 18 26497 26513 246 113031 113047 121988 122004 539312 n/a n/a Intron 1 CAGGAATGGAAAACCAA 40 26498 26514 247 113032 113048 121989 122005 539313 n/a n/a Intron 1 TCAGGAATGGAAAACCA 49 26499 26515 248 113033 113049 121990 122006 539314 n/a n/a Intron 1 TCTCCATTAGGGTTCTG 87 50948 50964 249 539315 n/a n/a Intron 1 TTCTCCATTAGGGTTCT 57 50949 50965 250 539316 n/a n/a Intron 1 GTTCTCCATTAGGGTTC 73 50950 50966 251 539317 n/a n/a Intron 1 AGGTTGGCAGACAGACA 73 53466 53482 252 539318 n/a n/a Intron 1 CAGGTTGGCAGACAGAC 84 53467 53483 253 539319 n/a n/a Intron 1 GCAGGTTGGCAGACAGA 85 53468 53484 254 539320 n/a n/a Intron 1 CTTCTTGTGAGCTGGCT 87 64884 64900 255 539321 n/a n/a Intron 1 TCTTCTTGTGAGCTGGC 89 64885 64901 256 539322 n/a n/a Intron 1 GTCTTCTTGTGAGCTGG 87 64886 64902 257 539323 n/a n/a Intron 1 AGTCTTCTTGTGAGCTG 70 64887 64903 258 539324 n/a n/a Intron 1 TCTTCCACTCACATCCA 65 65990 66006 259 539325 n/a n/a Intron 1 CTCTTCCACTCACATCC 78 65991 66007 260 539326 n/a n/a Intron 1 TCTCTTCCACTCACATC 68 65992 66008 261 539327 n/a n/a Intron 1 GTCTCTTCCACTCACAT 74 65993 66009 262 539328 n/a n/a Intron 1 ATAGATTTTGACTTCCC 57 72107 72123 263 539329 n/a n/a Intron 1 CATAGATTTTGACTTCC 35 72108 72124 264 539330 n/a n/a Intron 1 GCATAGATTTTGACTTC 53 72109 72125 265 539331 n/a n/a Intron 1 AAAATGTCAACAGTGCA 86 80639 80655 266 539332 n/a n/a Intron 1 CAAAATGTCAACAGTGC 73 80640 80656 267 539333 n/a n/a Intron 1 CCAAAATGTCAACAGTG 34 80641 80657 268 539334 n/a n/a Intron 1 CCCAAAATGTCAACAGT 66 80642 80658 269 539335 n/a n/a Intron 1 CATGACTATGTTCTGGC 67 125594 125610 270 539336 n/a n/a Intron 1 ACATGACTATGTTCTGG 42 125595 125611 271 539337 n/a n/a Intron 1 CACATGACTATGTTCTG 29 125596 125612 272 539338 n/a n/a Intron 2 GAATTCTGAGCTCTGGA 77 145429 145445 273 539339 n/a n/a Intron 2 TGAATTCTGAGCTCTGG 84 145430 145446 274 539340 n/a n/a Intron 2 CTGAATTCTGAGCTCTG 80 145431 145447 275 539341 n/a n/a Intron 2 CCTGAATTCTGAGCTCT 84 145432 145448 276 539342 n/a n/a Intron 2 GCCTGAATTCTGAGCTC 84 145433 145449 277 539343 n/a n/a Intron 2 AGCCTGAATTCTGAGCT 80 145434 145450 278 539344 n/a n/a Intron 2 ATATTGTAATTCTTGGT 0 148059 148075 279 539345 n/a n/a Intron 2 GATATTGTAATTCTTGG 20 148060 148076 280 539346 n/a n/a Intron 2 TGATATTGTAATTCTTG 13 148061 148077 281 539347 n/a n/a Intron 2 CTGATATTGTAATTCTT 8 148062 148078 282 539348 n/a n/a Intron 2 CCTGATATTGTAATTCT 67 148063 148079 283 539349 n/a n/a Intron 2 GCCTGATATTGTAATTC 73 148064 148080 284 539350 n/a n/a Intron 2 TGCCTGATATTGTAATT 32 148065 148081 285 539351 n/a n/a Intron 2 AATTATGTGCTTTGCCT 58 148907 148923 286 539352 n/a n/a Intron 2 CAATTATGTGCTTTGCC 82 148908 148924 287 539353 n/a n/a Intron 2 TCAATTATGTGCTTTGC 68 148909 148925 288 539354 n/a n/a Intron 2 GTCAATTATGTGCTTTG 80 148910 148926 289 539355 n/a n/a Intron 2 GCCATCACCAAACACCA 94 150972 150988 290 539356 n/a n/a Intron 2 TGCCATCACCAAACACC 84 150973 150989 291 539357 n/a n/a Intron 2 TTGCCATCACCAAACAC 74 150974 150990 292 539358 n/a n/a Intron 2 TGGTGACTCTGCCTGAT 85 151387 151403 293 539359 n/a n/a Intron 2 CTGGTGACTCTGCCTGA 86 151388 151404 294

TABLE 127 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 NO: 2 ISIS Start Stop SEQ ID NO Sequence % inhibition Site Site NO 523561 TATTTCAGAAAGACTTTCTG 11 10373 10392 295 523562 AGGAAAAAATCAAGGAGTTA 8 11173 11192 296 523563 TATTTACTGAACACCTATTC 12 11973 11992 297 523564 GCCCATGAAGGCCACTCTTC 70 12780 12799 298 523565 ACCTATAAATAAAGTGAGGA 0 13581 13600 299 523566 GTTTCATAACCTGCTAATAA 40 14451 14470 300 523567 ATGTGCCTTACAGTTATCAG 36 15251 15270 301 523568 TTCTGAATTTAGAATTATAG 0 16051 16070 302 523569 GTTTATAATCTAGCAGTTAC 26 17130 17149 303 523570 GATGGTCTTTCATGAATCAA 62 17930 17949 304 523571 CATGTACCCAAACCAACACT 65 18730 18749 305 523572 TAAAATACAGCCTACATCAT 0 19637 19656 306 523573 CCATCACTACAACAAACTCA 39 20451 20470 307 523574 ATCTGAAATGATCCCCTTTC 33 21283 21302 308 523575 TGTTGCCCCTCCAAAAAGAC 12 22144 22163 309 523576 ATTAAAATTTTAAATGATGT 0 22944 22963 310 523577 CTCAGGAATGGAAAACCAAA 71 26497 26516 311 113031 113050 121988 122007 523578 AAAATTCTAGAAGATAACAT 0 27838 27857 312 523579 CTAGAAGTCCTAGCCAGAGT 2 28748 28767 313 523580 AACCGATATCACAGAAATAC 0 29548 29567 314 523581 AAGATAGACAGTAACATAAT 0 30348 30367 315 523582 GCACTACAAGAACTGCTTAA 40 31172 31191 316 523583 TTTCCAGACAAAGAATTCAG 6 31978 31997 317 523584 GTAGACAGCCTTTCTGGAAC 20 32827 32846 318 523585 CATCCTACATAGTGGCTGTG 47 33635 33654 319 523586 CAGAACAGTGTGTGGAGACT 8 34452 34471 320 523587 AGCTTTAAAAATACCTCTGC 52 35466 35485 321 523588 CCCAGGTACTTGCTCTCAGA 22 36266 36285 322 523589 TTACACCTGATTCTAGAAAT 30 37066 37085 323 523590 CTTTTCTCTACAACCTCACA 34 38094 38113 324 523591 TAGTAGTTTGAATTTCAAAG 1 38909 38928 325 523592 ATACAAAATTACTGCAGTCA 60 39719 39738 326 523593 GCCACTGCCAAAAAGGAGGA 30 40519 40538 327 523594 TGACAGAAACAGAGCTATGA 33 41342 41361 328 523595 ATCTTTTAGTATGAACCTTA 65 42142 42161 329 523596 AGTTATGTGACTCTGAGCAC 63 43173 43192 330 523597 ACTATGCCCTAGTTACTTCT 29 43973 43992 331 523598 TATAGTGGAAGTGATAGATC 0 44812 44831 332 523599 TGTTTTCTGAAATGGAATGT 0 45733 45752 333 523600 GCTGTAAATGTAATGAGTGT 34 46553 46572 334 523601 GAGAGAAGCCATGGCCCTAG 20 47392 47411 335 523602 CTCTCTTTCCCAGAACAAGA 32 48210 48229 336 523603 TCCAAAATGTCCAGTATAAT 33 50072 50091 337 523604 GTTCTCCATTAGGGTTCTGG 74 50947 50966 338 523605 TTAGTCACCCATCCACCACT 41 51747 51766 339 523606 CATGAATTCACCGAGTTAGG 51 52573 52592 340 523607 AGCAGGTTGGCAGACAGACA 62 53466 53485 341 523608 GAAAGACTTAAATTTTCACA 0 54306 54325 342 523609 TAGTAGAGGAAAAGGAGAAT 0 55730 55749 343 523610 AAACAGGGTCTGGAGTGGAC 3 61243 61262 344 523611 CAAGCTGATAATTAAAAAGA 0 62462 62481 345 523612 ATAAAGATACATTTTCTGGG 8 63277 63296 346 523613 CAGGATTCTTCCTGCCTGGC 47 64085 64104 347 523614 AAGTCTTCTTGTGAGCTGGC 71 64885 64904 348 523615 CTCTTCCACTCACATCCATT 63 65988 66007 349 523616 CCTATATCAGAAGACAAATG 5 66806 66825 350 523617 TCAAAACCCTGCCAAGGTAC 44 67662 67681 351 523618 TCATATTCTACTTCTGTTTA 11 68462 68481 352 523619 CATTCCAGTGTTTCAGTAAG 13 69262 69281 353 523620 GGCCTGGAATTAATCCTCAG 49 70114 70133 354 523621 AATGCCCTCTCCCTGTGCCT 48 70925 70944 355 523622 TTTATAATCAACCTTTGCTA 9 71741 71760 356 523623 ATATAACTACTTAAAATAAT 0 72541 72560 357 523624 TTAGCCAGGATATGGTTGCC 50 73350 73369 358 523625 CTACCTCCATCAAAGAAAAT 0 74190 74209 359 523626 GCATGCATAGATAAGTTTGA 20 74990 75009 360 523627 ATGAGAGTAAATGGATTTTC 10 75790 75809 361 523628 TTGGCAATCCTTGCTTAAAA 34 76598 76617 362 523629 GAATTAAGCCAGACTTATTT 3 77398 77417 363 523630 GGCTAAGTATTTGTAGCAGT 55 78198 78217 364 523631 TTGCCTGTGTGCAACTGGCG 0 79005 79024 365 523632 GTGGCCTTAGTAGGCCAGCT 0 79827 79846 366 523633 CCCAAAATGTCAACAGTGCA 70 80639 80658 367 523634 TTAAGCCTTCAATTTGAAAA 0 81455 81474 368 523635 TGCTCAGAAGGTTGAGCATA 0 82261 82280 369 523636 TTAATGCTTTCCCAAAGCTC 35 83061 83080 370 523637 AAAAGACTTCATACCTTTAC 52 83884 83903 371

TABLE 128 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 NO: 2 ISIS Start Stop SEQ ID NO Sequence % inhibition Site Site NO 532146 GGCCCCCTGGCACAACAGGA 60 3097 3116 372 532147 TCTAGGGTGATTCAGGTGGA 62 4537 4556 373 532148 CTTAGATTAATGCAAAACAA 25 4875 4894 374 532149 AGGCAGAGGAGGGTGGAACC 34 6246 6265 375 532150 AGTCTAATGAGATCTGATGG 76 6499 6518 376 532151 GCTGAAATGAGTTAAGACTT 89 6737 6756 377 532152 ACTTTGGACTGTGGATTTTT 78 6765 6784 378 532153 GCATATTTACACAATGCCTG 84 6871 6890 379 532154 GGAAATGCCTGGATGTCCAG 27 7241 7260 380 532155 CTGCTGATTTTGGAATGGAG 68 10660 10679 381 532156 ACTGAACACCTATTCTATGG 51 11968 11987 382 532157 TTTACTGAACACCTATTCTA 23 11971 11990 383 532158 CCCTCAAATTATCCACAAAC 89 12053 12072 384 532159 CTTCTAAATGTTTCCAAGGC 63 12186 12205 385 532160 TTACATCCTGTAGGCTAATT 82 12469 12488 386 532161 CCACTAGCCTGGCCAGACTT 73 12487 12506 387 532162 CTGGTAGATGATCTCAAGTT 84 13351 13370 388 532163 AAAGAATTGAGTTATAAATC 23 13670 13689 389 532164 AACTCATCTCTGGCCAGCAG 89 14361 14380 390 532165 CAACATCATTGTATTTTCTG 33 14965 14984 391 532166 TCTTAGCTTACCAATGAGGA 81 15085 15104 392 532167 TTCCCAGAGCCAAAGCTCAA 77 15982 16001 393 532168 TTTGGCCAATCCCAGCTTAT 59 16253 16272 394 532169 GTTTGCAAATCTTCATTCAC 71 16447 16466 395 532170 CAATAGTCCCTGAGGCTTGG 74 16476 16495 396 532171 TTTCCCCAGATTAAATGCCC 85 17650 17669 397 532172 TTCAATAATGCAGTTATTAT 0 18308 18327 398 532173 AAATTCTTGGGCTTAAGCAC 69 18638 18657 399 532174 ACATGTACCCAAACCAACAC 71 18731 18750 91 532175 TGATCCAAATTCAGTACCTA 82 18752 18771 400 532176 GATGATCCAAATTCAGTACC 54 18754 18773 401 532177 CAATATTCATCTTTATATTC 25 19106 19125 402 532178 ATTGCTCTTAAGATAAGTAA 41 19661 19680 403 532179 CAGCTCCCTGAATATCTCTT 74 19783 19802 404 532180 ACTTCACAAATATATTATAA 0 19885 19904 405 532181 GTACAGTCAACTTTACTTCA 89 19899 19918 406 532182 CAATTCCCACTCTTGTCAAC 55 20288 20307 407 532183 TCAACTGCTTTCTGGAGCAG 66 21215 21234 408 532184 ACTGCTGAGCACCTCCAAAA 73 21454 21473 409 532185 CTTAGATTCCTGGTTTATCA 78 21587 21606 410 532186 AGTTATATCAATATTCTCCC 88 21820 21839 411 532187 TATACCATCTTCCCCATAAA 32 22038 22057 412 532188 GGCTTTCTTTAGCAATAGTT 86 22518 22537 413 532189 TACCAGGGATGTAGGTTTAC 82 29050 29069 414 532190 TCACAGCTGAATTCTATCTG 80 29323 29342 415 532191 GGAGATGGACAAATTCCTGC 77 29470 29489 416 532192 CTAGACATGTCATCAAGACA 19 30294 30313 417 532193 CAAATTAATAAAACAATTAC 10 30385 30404 418 532194 TATTCTTATATCAGACAAAA 30 30532 30551 419 532195 TCAAGGGATCCCTGCCATTC 32 32361 32380 420 532196 CGTCAAGGGATCCCTGCCAT 47 32363 32382 421 532197 GGCACTCCCAGTCTCCAGCT 83 34138 34157 422 532198 TTTCTCCAGCAGAAGTGTCA 60 34845 34864 423 532199 AAGTCCTCTTCCGCCTCCCT 82 36023 36042 424 532200 GGAATTTACCAAAAACAGTT 63 36721 36740 425 532201 AGTTAGGTATTGTCCATTTT 74 37032 37051 426 532202 ACATGGGTATCTTCTAGGAA 77 37111 37130 427 532203 TCAGTTTCAGAGAGACAAAA 41 37276 37295 428 532204 TTTGCCAGGTCCTATGTCGA 69 37656 37675 429 532205 ATTCCCTTTTCTCTACAACC 70 38099 38118 430 532206 ATGATAAGAGCCAAGATTTG 13 38994 39013 431 532207 GAAAAAAGGTCCACTGTGGT 49 40356 40375 432 532208 CCTGTCCTGGAATAGTTTCA 49 41164 41183 433 532209 TAGAAAAGTAAATAAGGAAT 15 41501 41520 434 532210 TTATAAAACTATGCAATAGG 0 41889 41908 435 532211 TTATTTCATATTTCCAGAAA 0 42675 42694 436 532212 CATGAATTACAGCTAAAGAT 20 42741 42760 437 532213 TTGCATGTATGTGTTTCTGA 62 43518 43537 438 532214 TCAATCTCTTTATACCCTTA 75 43765 43784 439 532215 TCTTCAATCTCTTTATACCC 58 43768 43787 440 532216 CTATGCCCTAGTTACTTCTA 47 43972 43991 441 532217 AAAGAGAATCTCTTCCTTTT 27 44070 44089 442 532218 TCATTAAAGATTATTATAAC 0 44222 44241 443 532219 TTTGGATGAGTGGAAGGCTA 0 44528 44547 444 532220 GGAAATGGCCTTTTTCCTTA 72 45400 45419 445 532221 GGAGAAGCCCTCTGCCTGTA 60 46477 46496 446 532222 AAACCATATTGTCCACCAGA 84 46510 46529 447

TABLE 129 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 SEQ ISIS % Start Stop ID NO Sequence inhibition Site Site NO 532223 CTCAAACCATATTGTCCACC 90 46513 46532 448 532224 GTGTAAATAGTGACTTGTAC 76 50123 50142 449 532225 TGAGGCACAGGAAAGTTAAC 52 50719 50738 450 532226 AGCTATAGTTCTCCATTAGG 74 50954 50973 451 532227 TTACTTGCTGACTAAGCCAT 69 51071 51090 452 532228 GTTTGTCAACTCAACATCAA 73 51215 51234 453 532229 GACTATTTGTATATATATAC 33 51491 51510 454 532230 ATGACTATTTGTATATATAT 11 51493 51512 455 532231 ACTCTTCCTTATATTTGCTC 76 51778 51797 456 532232 ATACACTGACTTTTAACATT 67 52039 52058 457 532233 CTTAGAAACAGTAGTTTCAT 42 52124 52143 458 532234 CTGAGCTTTGCCTTAAGAAT 79 52633 52652 459 532235 CACCAGACAGCAGGTAGAGC 81 53540 53559 460 532236 GAGATGGAGTAGAAGGCAAA 43 55926 55945 461 532237 TAGGAAAGGAAGAATACACT 33 63881 63900 462 532238 TAGACCAGGAAGGGTGAGAG 27 64376 64395 463 532239 AAGTTGGATCTGGCATGCAT 64 64574 64593 464 532240 AAAGTTGGATCTGGCATGCA 70 64575 64594 465 532241 CCATAACTCTTCTAACTGGG 84 64643 64662 466 532242 ATATTAAAGTTTGAGAACTA 37 65080 65099 467 532243 CTTAACTACAAAATGCTGGA 71 66164 66183 468 532244 TGAGCAGCTGTCCTCAGTTC 43 67061 67080 469 532245 GAGTTCATAAAAGTTTTACT 26 67251 67270 470 532246 CTATCCACACCATTCCATAA 73 69203 69222 471 532247 AACATCTAAGTAATGCAAAC 58 69223 69242 472 532248 TTTGCATTCAAAGCCCTGGG 91 69565 69584 473 532249 TCCATATTATAGGCTATGAT 73 69889 69908 474 532250 ATTTTATGATAATGTAAAAC 27 69942 69961 475 532251 GAGATCACATTTTCTGAGTA 50 70352 70371 476 532252 ACCTCCCTAGGATTACCTCA 56 71617 71636 477 532253 AAAATCTGATTTATAATCAA 40 71750 71769 478 532254 AGCATAGATTTTGACTTCCC 92 72107 72126 479 532255 AAAGTCATATACACAGGTCT 53 72584 72603 480 532256 CTCATAGCAAATTCCCAGAA 66 73689 73708 481 532257 CAACATGGAGGCTAGCATGT 55 74112 74131 482 532258 AGACTAAGTGGCCTGAATGT 52 74317 74336 483 532259 ACCTACCATGTCACTCTCAA 61 74418 74437 484 532260 AACTTTCTTGTGTTTTATCA 9 75511 75530 485 532261 TTTGCAAGACAAAGAAATGA 31 75915 75934 486 532262 CATGCAAAGTGTTCCTCTTC 63 76024 76043 487 532263 AGTGCTTTGCTTTCTCTTAT 79 76047 76066 488 532264 GAACAAGAAACACTTGGTAA 44 76555 76574 489 532265 AGTGTTCCAATTAAATGGCA 34 76643 76662 490 532266 AAACAATGCCCTTGTAGTGA 57 76703 76722 491 532267 TATTCTAGGTTTTGAGGTGA 60 76752 76771 492 532268 ATATTCTAGGTTTTGAGGTG 24 76753 76772 493 532269 GTTTTCCATTCTTTAAGAAA 41 76896 76915 494 532270 AGCAATCCATTGATTGTATG 59 77044 77063 495 532271 AATTATGGCAAAATGGAAAA 37 77076 77095 496 532272 ACATTTGCTTATGAGACTAT 62 77638 77657 497 532273 GCAGAGATAATCCTATGATG 42 77841 77860 498 532274 TCCATCTGTTACCTCTCTGT 77 78122 78141 499 532275 TTTGCCTGAAGGGCAGAACC 40 79478 79497 500 532276 GAAAAAATCAGATTTTCACA 0 79664 79683 501 532277 AACTTAATTTAATCATTTCT 0 79959 79978 502 532278 TTTGGTTGTCATGAGTTGAG 67 80756 80775 503 532279 TTCCATCTCTAGGGCACTTT 74 80900 80919 504 532280 AGAGCTTATTTTCAAAATTC 36 80920 80939 505 532281 ATAAAGAGCAAACAAACATA 42 81524 81543 506 532282 TATAAATTCCTTGGTCTGAT 33 82835 82854 507 532283 AAAATATAAATTCCTTGGTC 13 82839 82858 508 532284 TTTTATAACAGCCTCTGACA 38 82959 82978 509 532285 AAAAGACCATGTTGCTTATT 72 83179 83198 510 532286 ATAGTCAGTCAGAATGTGGT 72 83330 83349 511 532287 TGCCTTAGCTTGGAAAAGAC 78 83897 83916 512 532288 AGGGCTAGCTGATGCCTCTC 69 84026 84045 513 532289 TTGGACTGGGCTCAAACAGA 72 84381 84400 514 532290 AAAGTCAGGCTAGAGGGACT 49 85713 85732 515 532291 TCCTTGTTTTCTTGTAATGA 50 85945 85964 516 532292 ACACCAGAGGAAGGAAATCA 44 86554 86573 517 532293 GATGTACACCATTTTGAATT 15 86629 86648 518 532294 TGCTCTGGCCTAGCCTATGT 62 86901 86920 519 532295 CAGAGGTGTCTCCCAAGAAA 60 89940 89959 520 532296 AAAGAGAATGGATCAAAGCT 36 91930 91949 521 532297 GATTTGCAGAACAAATCTTG 37 93332 93351 522 532298 TGGTTATGAAGGTTGGACCA 52 94839 94858 523 532299 TGGCTAATTAATGGGCAATT 63 95292 95311 524

TABLE 130 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 SEQ ISIS % Start Stop ID NO Sequence inhibition Site Site NO 532300 CTGTGCCATATTGCCTCTAA 87 95471 95490 525 532301 GATTTCAACCAGCTCACCTG 48 95510 95529 526 532302 GCAAAAGGGAACCCTGAAGC 71 95564 95583 527 532303 CTAAGTGTTATAACAAACAC 43 96137 96156 528 532304 GTCCATTGGTATAAAACTCA 84 96282 96301 529 532305 TTTCAATACAATAAGATTTA 34 96793 96812 530 532306 GTCCTTAGACCCCTCAATGG 62 96987 97006 531 532307 GAGGATTTATTCATCTAGGC 68 97806 97825 532 532308 CAGTGGGAGGATCAGATATC 46 97870 97889 533 532309 ATCCCATCCAGCAGCTGGAC 67 98132 98151 534 532310 AACTTGGGATGAGTTACTGA 56 98653 98672 535 532311 GAAGGCTACCTAAAAGAAAT 43 98810 98829 536 532312 AAAGAAATATTCACAACATT 39 99096 99115 537 532313 ATGCTTATACTGCTGCTGTA 69 99791 99810 538 532314 TCCTCACTTCAATCACCTTT 70 99819 99838 539 532315 CTCTTTCTTCATAAATAAGT 33 100809 100828 540 532316 TGGTAATCTGTGTCCCTTTA 96 101242 101261 541 532317 TAATAAAAAAGTTTGAAACA 41 102549 102568 542 532318 GGTGGTGGCAAGAGAAAAAT 56 103015 103034 543 532319 CAAAAGGCCCTTTTTACATG 28 103034 103053 544 532320 ACTCTACTGGTACCAATTTA 31 103173 103192 545 532321 TCTGAACTTTTATGCTCTGT 76 103606 103625 546 532322 AACTTTTGCCTGGGCATCCA 16 104067 104086 547 532323 TGACTCCATGTCTCACATCC 66 104392 104411 548 532324 TTACTTCCTAGATACAACAG 53 104541 104560 549 532325 CTGGCCCCCATGATTCAATT 44 104835 104854 550 532326 AAGACTGGCCCCCATGATTC 49 104839 104858 551 532327 TGTCACTGGTCTGTGTATTT 60 106233 106252 552 532328 ACAGAGTAGATTTAGCATAA 23 106980 106999 553 532329 TAAACAGGTGTACTATTACA 27 107030 107049 554 532330 GCTTTATCAACTAAGTTTAT 22 107716 107735 555 532331 CAGAACTTCTTTTAAAATTG 8 107763 107782 556 532332 GAATACAGACATACCTTGAA 25 108514 108533 557 532333 CCATGACAACAATTTCAGAG 58 109486 109505 558 532334 ACAAATAGCAATGAATGGGT 45 110878 110897 559 532335 CAACAAATAGCAATGAATGG 47 110880 110899 560 532336 GTACACAAATCAGTAGCTCT 72 115087 115106 561 532337 CTATGTCAAAAAGACTGAAA 4 116370 116389 562 532338 ATATACAGAACATTTCATCC 13 116743 116762 563 532339 AGAATAGATAAGAACTCACC 32 117195 117214 564 532340 AGGAAAGATACAGTCATTTT 5 117507 117526 565 532341 GCACAAAGAACACCTGGGAA 43 117781 117800 566 532342 CAAGAAGTCTGGGATTATGT 0 117938 117957 567 532343 GTTAGTTATTAAGCTAATCA 48 118245 118264 568 532344 AACCATTATTTATAGGCTAA 14 119127 119146 569 532345 CCAGAATGCGATCACTTCTT 76 120826 120845 570 532346 CCAGAAATTATCCTCCTCTC 70 121209 121228 571 532347 AGGGAAATGCAAATTAAAAC 20 122479 122498 572 532348 GCATCAAGATACAGAAAAAT 24 122751 122770 573 532349 GAATGTTTATGAGATTTTTC 0 123571 123590 574 532350 GCCAATTATATTGCCACATT 23 124413 124432 575 532351 ATACTTGCTTATGTAGAAAT 45 124589 124608 576 532352 TAATACTTGCTTATGTAGAA 3 124591 124610 577 532353 GAACACATGGCATTCTGATA 36 125178 125197 578 532354 CAGAATTTGCAGTATAAATC 0 126051 126070 579 532355 TATGTTTTGAAATCTTATTT 0 126157 126176 580 532356 ACTCACTGCTACCTCATTAA 11 126998 127017 581 532357 AAGCAGTGATAGGGTATCTG 59 127080 127099 582 532358 ATGAGGCCTATTACAATGGA 14 127170 127189 583 532359 CTGGAGTCTCATGAGGCCTA 53 127180 127199 584 532360 TGACTATCAGCCTTTTAATC 45 127663 127682 585 532361 TTCAGAGAACAACCTTTGAA 0 127959 127978 586 532362 AGCCATGTGTGATCTGATGT 53 128813 128832 587 532363 GAAATTTACTCCAAACTAGC 17 128992 129011 588 532364 AACATCCAGACCACCATCTA 35 130094 130113 589 532365 GTACCAAACCATTCATGCTC 56 131036 131055 590 532366 AGTACCAAACCATTCATGCT 24 131037 131056 591 532367 TTATAGAGCTTGAGATTGAC 7 132165 132184 592 532368 AGTCCATTATAGAGCTTGAG 58 132171 132190 593 532369 AACCATGAGATGCAATGCAG 40 132498 132517 594 532370 AGGATTGAGAATCGCTGATT 42 133168 133187 595 532371 TCTAAAGCATGGCCAGGATT 48 133182 133201 596 532372 GGGACTGAGTATTGATACTT 44 133222 133241 597 532373 AGAAGTAGGGTGTTCCAGAT 29 133523 133542 598 532374 AGAAATAGTCTTCCTACTAA 0 133547 133566 599 532375 GCCTCCTTTAAGCTTCTATG 22 134240 134259 600 532376 GGCCTGCCTTTACTTTCCCA 36 134598 134617 601

TABLE 131 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 1 and 2 of SEQ ID NO: 2 SEQ SEQ ID ID SEQ SEQ NO: 1 NO: 1 ID NO: ID NO: SEQ ISIS Start Stop Target % 2 Start 2 Stop ID NO Site Site Sequence region inhibition Site Site NO 523638 n/a n/a ACCTCAGTGGACTCTTTCCA Intron 1 4 84684 84703 602 523639 n/a n/a CAAACCTAAGTTCAAGTCCT Intron 1 62 85523 85542 603 523640 n/a n/a AGTTTCACTTCTTGAATCAA Intron 1 38 86373 86392 604 523641 n/a n/a AAGATCAAATGAGGTCAAGG Intron 1 30 87181 87200 605 523642 n/a n/a TAGATACAAATTTCATCACA Intron 1 23 88063 88082 606 523643 n/a n/a ATTCCTAAAATAGGAGCAGG Intron 1 45 88870 88889 607 523644 n/a n/a TTTTTATGTTGTATAAGATA Intron 1 0 89670 89689 608 523645 n/a n/a GTTCAGCCAATACATGAGTA Intron 1 48 90473 90492 609 523646 n/a n/a CCAGAGGGAGTTCATTACCA Intron 1 62 91273 91292 610 523647 n/a n/a TCTCTCTAATTCAACCTTAT Intron 1 44 92107 92126 611 523648 n/a n/a ATAATCCTCAGACCTCTTTA Intron 1 29 92925 92944 612 523649 n/a n/a CACTGTGGCAGAATTCCAAG Intron 1 28 93762 93781 613 523650 n/a n/a ACACCTTGGTGCCTAGAAGC Intron 1 54 94581 94600 614 523651 n/a n/a GTAGCAATGACACCTAAGAA Intron 1 58 95394 95413 615 523652 n/a n/a TTTAAAATAATAAATGCTTA Intron 1 0 96194 96213 616 523653 n/a n/a TCATTTGGTCCTTAGACCCC Intron 1 27 96994 97013 617 523654 n/a n/a TTATTCATCTAGGCCGAGTG Intron 1 57 97800 97819 618 523655 n/a n/a TTGCAGAATCTTCTCTTTGG Intron 1 65 98627 98646 619 523656 n/a n/a ACCATAAAGCTATGCCATAA Intron 1 63 99481 99500 620 523657 n/a n/a GGCAAGGAGCACAATAGGAC Intron 1 20 100281 100300 621 523658 n/a n/a ACCCAAAAAGTTGAGCTGAG Intron 1 66 101081 101100 622 523659 n/a n/a TAGATTTTCAGACTCTTTCT Intron 1 46 101887 101906 623 523660 n/a n/a AATTTCAATATTGTTGTGTT Intron 1 0 102760 102779 624 523661 n/a n/a ATGCTTTTAATGGCACCCAA Intron 1 69 103569 103588 625 523662 n/a n/a CATGTCTCACATCCAGGTCA Intron 1 37 104386 104405 626 523663 n/a n/a TTCACTGGAGTAGACTTTTA Intron 1 45 105255 105274 627 523664 n/a n/a CTTATAAGGGAGGTCTGGTA Intron 1 41 106147 106166 628 523665 n/a n/a GCATAATTCTTAAGGGCCCT Intron 1 71 106966 106985 629 523666 n/a n/a CCACAGAACTTCTTTTAAAA Intron 1 27 107766 107785 630 523667 n/a n/a GGTGACCATGATTTTAACAA Intron 1 25 108566 108585 631 523668 n/a n/a AACAGCTGCATGACAATTTT Intron 1 50 109382 109401 632 523669 n/a n/a AGAAACAGAATCAGTGACTT Intron 1 44 110403 110422 633 523670 n/a n/a CAGATTCCAGAGAAAAGCCA Intron 1 14 111203 111222 634 523671 n/a n/a TGTGAGAAGAACTCTATCAC Intron 1 12 112030 112049 635 523672 n/a n/a CTCACAAATCACCACTAAAG Intron 1 31 112842 112861 636 523673 n/a n/a CAACGAGTGGATAAAGAAAC Intron 1 28 113646 113665 637 523674 n/a n/a ATAAAACTGGATCCTCATCT Intron 1 13 114446 114465 638 523675 n/a n/a ATTAAAACTCTCAGCAAAAT Intron 1 0 115450 115469 639 523676 n/a n/a AAAGACTGAAAGAACACAAA Intron 1 0 116361 116380 640 523677 n/a n/a TATCTGCTGCCTTCAGGAGA Intron 1 0 117168 117187 641 523678 n/a n/a TTTGAATTAACCCAATTCAA Intron 1 0 117999 118018 642 523679 n/a n/a TCTTAATTTACAACAGAGGA Intron 1 25 118821 118840 643 523680 n/a n/a AGAAAAGTGACAGGCTTCCC Intron 1 31 119659 119678 644 523681 n/a n/a ATGTTCCTTGAAGATCCCAA Intron 1 37 120478 120497 645 523682 n/a n/a ATGAATAACACTTGCCACAA Intron 1 0 121379 121398 646 523683 n/a n/a GTATGTTTATCACAGCACAG Intron 1 56 122180 122199 647 523684 n/a n/a AAACACTGCAATATTAGGTT Intron 1 34 123031 123050 648 523685 n/a n/a GATTGGTGCTTTTCAAACTG Intron 1 39 123936 123955 649 523686 n/a n/a ATTTGTAAGACAAACATGAA Intron 1 9 124764 124783 650 523687 n/a n/a TCACATGACTATGTTCTGGC Intron 1 72 125594 125613 651 523688 n/a n/a AGTCCTGTCCACACTATTAA Intron 1 6 126415 126434 652 523689 n/a n/a CTGGGCTCTGCCTGCTGAAC Intron 1 17 127217 127236 653 523690 n/a n/a AAAACCCTTAAGTATTTCCT Intron 1 12 128054 128073 654 523691 n/a n/a CTCTGTTTCAAACCCCCCAG Intron 1 21 128854 128873 655 523692 n/a n/a GGACAGAACACCAATCACAA Intron 1 18 129654 129673 656 523693 n/a n/a ACCTACCCTTCAAAGTCACG Intron 1 0 130486 130505 657 523694 n/a n/a TTCAGTTCCCAGGAGGCTTA Intron 1 5 131286 131305 658 523695 n/a n/a TTTTGCAATGTCTAGCAATT Intron 1 0 132086 132105 659 523696 n/a n/a ATTAAGATCAGAAAATATTA Intron 1 0 132953 132972 660 523697 n/a n/a TTAATGAGATATTTTGCACC Intron 1 34 133858 133877 661 523698 n/a n/a GAGAGGTTAAGTAAATCTCC Intron 1 0 134678 134697 662 523699 n/a n/a CAGACTCAAATTTGAAAATT Intron 1 14 135500 135519 663 523700 n/a n/a GATAAGGCAATAATACAGCC Intron 1 1 136306 136325 664 523701 n/a n/a ATCATTTGCCAATTTCTGTG Intron 1 28 137133 137152 665 523702 n/a n/a CAAGAAGAAAAGATGCAAAA Intron 1 0 138035 138054 666 523703 n/a n/a AATTTATTTCCTTCCTATGA Intron 1 0 138857 138876 667 523704 n/a n/a TTTTGGAAATGTGAGAAACG Intron 1 0 139771 139790 668 523705 n/a n/a AAACACATGAGAAAAGATGA Intron 1 0 140593 140612 669 523706 n/a n/a TGTTGGCTCAGTGGGAATGA Intron 1 0 141412 141431 670 523707 n/a n/a TGAACAGGTTTGCATTTCTC Intron 1 42 142229 142248 671 523708 n/a n/a TCCTAGGTGAACAGGCTATG Intron 1 38 143029 143048 672 523709 n/a n/a CCCTAATCAGGCTGAAATAA Intron 1 0 143829 143848 673 523710 n/a n/a AGGGCCAGTAAGGTTTGCTT Intron 1 12 144631 144650 674 523711 n/a n/a AGCCTGAATTCTGAGCTCTG Intron 2 88 145431 145450 675 523712 n/a n/a AGAGGATCTCAGCTGCAATT Intron 2 71 146238 146257 676 523713 n/a n/a GAAAATCCCTGCTCAAGTGC Intron 2 67 147262 147281 677 523714 n/a n/a TGCCTGATATTGTAATTCTT Intron 2 90 148062 148081 678

TABLE 132 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 1 and 2 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 SEQ ISIS Target % Start Stop ID NO Sequence Region inhibition Site Site NO 532377 CTCATACAGTGAAGTCTTCA Intron 1 73 135431 135450 679 532378 CTCACTAAGCTTGATTCACT Intron 1 67 135818 135837 680 532379 GATACAGAAATCCCAGTGAC Intron 1 46 136111 136130 681 532380 TGTGCTTGGGTGTACAGGCA Intron 1 71 136282 136301 682 532381 TCAAGCACTTACATCATATG Intron 1 42 136377 136396 683 532382 AGGGTTAGTTATTACACTTA Intron 1 60 136576 136595 684 532383 AGGCTTCATGTGAGGTAACA Intron 1 58 136996 137015 685 532384 TGAAAGCTTAGTACAAGAAG Intron 1 51 138048 138067 686 532385 CTCTCCTCTTGGAGATCCAG Intron 1 58 138782 138801 687 532386 GCTGAGATTTCTCTCCTCTT Intron 1 78 138792 138811 688 532387 CTTTTGCTGAGATTTCTCTC Intron 1 58 138797 138816 689 532388 GAACATATGTCCATAGAATG Intron 1 57 141700 141719 690 532389 GAACAGGCTATGTAATCAAA Intron 1 68 143021 143040 691 532390 TTTTTATTACTGTGCAAACC Intron 1 41 143878 143897 692 532391 ACTGAGGGTGGAAATGGAAA Intron 2 23 145059 145078 693 532392 ATGCCATACTTTTCATTTCA Intron 2 87 146351 146370 694 532393 TCTTTAAAGATTTCCTATGC Intron 2 66 146367 146386 695 532394 TCACAATTAAATTATGTTTA Intron 2 47 149858 149877 696 532395 TTTGCCATCACCAAACACCA Intron 2 94 150972 150991 697 532396 TCAGAATGCTGAAGGATGGG Intron 2 70 152208 152227 698 532397 ACAATTGCAGGAGAGAACTG Intron 2 57 152296 152315 699 532398 GTTCAGTCACCTGGAAAGAG Intron 2 62 152549 152568 700 532399 CGGAGTTCAGTCACCTGGAA Intron 2 77 152553 152572 701 532400 AATCTAAAGTTCAATGTCCA Intron 2 77 152752 152771 702 532401 CCACCTTTGGGTGAATAGCA Intron 2 95 153921 153940 703 532402 CAACATCAAAAGTTTCCACC Intron 2 81 153936 153955 704 532403 AAGCTTCTATCAACCAACTG Intron 2 87 154093 154112 705 532404 ACCATTTTCTAATAATTCAC Intron 2 46 154502 154521 706 532405 ACCTGCACTTGGACAACTGA Intron 2 60 154727 154746 707 532406 GTCAGTGCTTTGGTGATGTA Intron 2 11 155283 155302 708 532407 TAGAAGCACAGGAACTAGAG Intron 2 68 155889 155908 709 532408 TTTAATTTTATTAGAAGCAC Intron 2 14 155900 155919 710 532409 GAGCAAGAATTAAGAAAATC Intron 2 29 155973 155992 711 532410 CTCTGCAGTCATGTACACAA Intron 2 93 156594 156613 712 532411 GCTTGGTTTGTCAATCCTTT Intron 2 95 156889 156908 713 532412 GTTCTCAAGCAGGAGCCATT Intron 2 70 157330 157349 714 532413 AGGGTGATCTTCCAAAACAA Intron 2 87 158612 158631 715 532414 TCTCCTATGCTTCCTTTAAT Intron 2 25 158813 158832 716 532415 GACATAAATATGTTCACTGA Intron 2 81 159216 159235 717 532416 TTACTGAGTGACAGTACAGT Intron 2 65 161588 161607 718 532417 CCAGGCACCAGCACAGGCAC Intron 2 47 161950 161969 719 532418 TTAATGTCAGTAGAAAGCTG Intron 2 0 162349 162368 720 532419 GCAGGTGGAAAGAAGATGTC Intron 2 50 162531 162550 721 532420 GCCAGGGTCTTTACAAAGTT Intron 2 93 162751 162770 722 532421 CATTACCTTTGTACATGTAC Intron 2 83 164839 164858 723 532422 GAAGCAACTTCTCTGAGGTC Intron 2 68 165040 165059 724 532423 GCCTGGCAAGAAGGGCCCTT Intron 2 56 165856 165875 725 532424 ACACATGTTTTTAAATTTAT Intron 2 21 166241 166260 726 532425 TCACAATGCACTAAAAGAAA Intron 2 53 168760 168779 727 532426 TCCCAATGACTTACTGTAGA Intron 2 78 169073 169092 728 532427 TAAGCATTTATGGAGGAATG Intron 2 46 169134 169153 729 532428 TGAGGTGGGTGGCCAACAGG Intron 2 66 170081 170100 730 532429 GTTTTTCATTTTGATTGCAG Intron 2 88 170158 170177 731 532430 AGCTCAAGTGTTTTTCATTT Intron 2 64 170167 170186 732 532431 CAATGTCACAGCTGTTTCCT Intron 2 62 170272 170291 733 532432 GAACTTTGGAGGCTTTTAGA Intron 2 55 170703 170722 734 532433 TGTATGCCCCAAACTCCCAT Intron 2 83 171431 171450 735 532434 ACACAAATAAGGGAATAATA Intron 2 24 171549 171568 736 532435 TAGTTCAGCCACTATGGAAA Intron 2 47 171926 171945 737 532436 CTCCAAATTCCAGTCCTAGG Intron 2 93 172746 172765 738 532437 AGTTGGCACTGCTATATCAG Intron 2 66 173668 173687 739 532438 GGCCTTAGATTGTAAGTTTT Intron 2 69 174122 174141 740 532439 TTTTAGTATTATTGTAGGAA Intron 2 16 174188 174207 741 532440 TTTCATTAATGAAACCTGAT Intron 2 39 174812 174831 742 532441 CCCTCAGCTGCCTCTTCAAT Intron 2 51 175014 175033 743 532442 TATTGTATCCTGGCCCCTAA Intron 2 68 175689 175708 744 532443 AGAACAAGAGCCTAGAAGTA Intron 2 35 176592 176611 745 532444 GTGACTATGTCACTGAATTT Intron 2 14 176918 176937 746 532445 GCCCTACCCAGCAGCCTGTG Intron 2 79 177540 177559 747 532446 CAAACATAAAGAGAGTTCCA Intron 2 79 177811 177830 748 532447 CTTTAAATGAAGTAGAGCTC Intron 2 0 178090 178109 749 532448 CTGTTCAAAGAATGCAGGCC Intron 2 70 178905 178924 750 532449 GTCTAGCCTAACAGAGATAT Intron 2 47 179137 179156 751 532450 AAAGAGTGATGTCTAGCCTA Intron 2 55 179147 179166 752 532451 CACTTCTTACTCCTTTGAGG Intron 2 50 179631 179650 753 532452 TTCCACAAGAAACTCAGTTT Intron 2 56 181514 181533 754 532453 AGAAATGCCAAAGATAGCTC Intron 2 56 182105 182124 755

TABLE 133 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 2 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 SEQ ISIS % Start Stop ID NO Sequence inhibition Site Site NO 533249 AGCAGAGGATCTCAGCTGCA 84 146241 146260 756 533250 AATCCCTGCTCAAGTGCTAC 75 147259 147278 757 533251 AAATCCCTGCTCAAGTGCTA 71 147260 147279 758 533252 AAAATCCCTGCTCAAGTGCT 73 147261 147280 759 533253 AGAAAATCCCTGCTCAAGTG 56 147263 147282 760 533254 AAGAAAATCCCTGCTCAAGT 58 147264 147283 761 533255 CAAGAAAATCCCTGCTCAAG 46 147265 147284 762 533256 CTGATATTGTAATTCTTGGT 91 148059 148078 763 533257 CCTGATATTGTAATTCTTGG 90 148060 148079 764 533258 GCCTGATATTGTAATTCTTG 94 148061 148080 765 533259 ATGCCTGATATTGTAATTCT 91 148063 148082 766 533260 AATGCCTGATATTGTAATTC 74 148064 148083 767 533261 CAATGCCTGATATTGTAATT 76 148065 148084 768 533262 AATTATGTGCTTTGCCTGCA 92 148904 148923 769 533263 CAATTATGTGCTTTGCCTGC 83 148905 148924 770 533264 TCAATTATGTGCTTTGCCTG 83 148906 148925 771 533265 TGTCAATTATGTGCTTTGCC 91 148908 148927 772 533266 ATGTCAATTATGTGCTTTGC 83 148909 148928 773 533267 GATGTCAATTATGTGCTTTG 74 148910 148929 774 533268 CTGGTGACTCTGCCTGATGA 77 151385 151404 775 533269 GCTGGTGACTCTGCCTGATG 87 151386 151405 776 533270 TGCTGGTGACTCTGCCTGAT 89 151387 151406 777 533271 GCTGCTGGTGACTCTGCCTG 94 151389 151408 778 533272 GGCTGCTGGTGACTCTGCCT 77 151390 151409 779 533273 TGGCTGCTGGTGACTCTGCC 82 151391 151410 780 533274 GCTGAAGGATGGGCATCCAG 85 152201 152220 781 533275 TGCTGAAGGATGGGCATCCA 85 152202 152221 782 533276 ATGCTGAAGGATGGGCATCC 78 152203 152222 783 533277 GAATGCTGAAGGATGGGCAT 66 152205 152224 784 533278 AGAATGCTGAAGGATGGGCA 81 152206 152225 785 533279 CAGAATGCTGAAGGATGGGC 85 152207 152226 786 533280 TCCAGTAGTCAATATTATTT 87 153001 153020 787 533281 ATCCAGTAGTCAATATTATT 85 153002 153021 788 533282 TATCCAGTAGTCAATATTAT 69 153003 153022 789 533283 GTTATCCAGTAGTCAATATT 77 153005 153024 790 533284 GGTTATCCAGTAGTCAATAT 85 153006 153025 791 533285 TGGTTATCCAGTAGTCAATA 86 153007 153026 792 533286 CAACTTGAGGACAATAAGAG 35 155591 155610 793 533287 TCAACTTGAGGACAATAAGA 62 155592 155611 794 533288 CTCAACTTGAGGACAATAAG 86 155593 155612 795 533289 AACTCAACTTGAGGACAATA 82 155595 155614 796 533290 TAACTCAACTTGAGGACAAT 66 155596 155615 797 533291 ATAACTCAACTTGAGGACAA 87 155597 155616 798 533292 CAGGAAGAAAGGAACCTTAG 77 156391 156410 799 533293 CCAGGAAGAAAGGAACCTTA 84 156392 156411 800 533294 ACCAGGAAGAAAGGAACCTT 86 156393 156412 801 533295 AGACCAGGAAGAAAGGAACC 74 156395 156414 802 533296 TAGACCAGGAAGAAAGGAAC 59 156396 156415 803 533297 ATAGACCAGGAAGAAAGGAA 65 156397 156416 804 533298 TACAATGCACAGGACACGCC 73 157198 157217 805 533299 CTACAATGCACAGGACACGC 85 157199 157218 806 533300 GCTACAATGCACAGGACACG 83 157200 157219 807 533301 ATGCTACAATGCACAGGACA 89 157202 157221 808 533302 TATGCTACAATGCACAGGAC 82 157203 157222 809 533303 ATATGCTACAATGCACAGGA 84 157204 157223 810 533304 CTGATATTTATTGCTGTACG 76 158006 158025 811 533305 CTCTGATATTTATTGCTGTA 80 158008 158027 812 533306 TCTCTGATATTTATTGCTGT 86 158009 158028 813 533307 GTCTCTGATATTTATTGCTG 80 158010 158029 814 533308 CCAGAAGAATTACCCATGCA 85 165550 165569 815 533309 TCCAGAAGAATTACCCATGC 84 165551 165570 816 533310 TTCCAGAAGAATTACCCATG 81 165552 165571 817 533311 TCTTCCAGAAGAATTACCCA 58 165554 165573 818 533312 ATCTTCCAGAAGAATTACCC 64 165555 165574 819 533313 CATCTTCCAGAAGAATTACC 58 165556 165575 820 533314 TTTCTGCAGTATCCTAGCCT 78 166350 166369 821 533315 GTTTCTGCAGTATCCTAGCC 88 166351 166370 822 533316 AGTTTCTGCAGTATCCTAGC 86 166352 166371 823 533317 TCAGTTTCTGCAGTATCCTA 88 166354 166373 824 533318 TTCAGTTTCTGCAGTATCCT 87 166355 166374 825 533319 TTTCAGTTTCTGCAGTATCC 80 166356 166375 826 533320 GTTTCCATTTTCTTGATTCC 70 169601 169620 827 533321 TGTTTCCATTTTCTTGATTC 54 169602 169621 828 533322 GTGTTTCCATTTTCTTGATT 55 169603 169622 829 533323 TGGTGTTTCCATTTTCTTGA 73 169605 169624 830 533324 ATGGTGTTTCCATTTTCTTG 76 169606 169625 831 533325 AATGGTGTTTCCATTTTCTT 78 169607 169626 832

TABLE 134 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 ISIS Target % Start Stop SEQ ID NO Sequence region inhibition Site Site NO 533326 AACCCATTTCATCCATTTAA Intron 2 93 175369 175388 833 533327 GAACCCATTTCATCCATTTA Intron 2 83 175370 175389 834 533328 GGAACCCATTTCATCCATTT Intron 2 92 175371 175390 835 533329 TAGGAACCCATTTCATCCAT Intron 2 91 175373 175392 836 533330 GTAGGAACCCATTTCATCCA Intron 2 95 175374 175393 837 533331 GGTAGGAACCCATTTCATCC Intron 2 92 175375 175394 838 533332 TGAGGGATTGCCTCAGTAGC Intron 2 66 179616 179635 839 533333 TTGAGGGATTGCCTCAGTAG Intron 2 72 179617 179636 840 533334 TTTGAGGGATTGCCTCAGTA Intron 2 67 179618 179637 841 533335 CCTTTGAGGGATTGCCTCAG Intron 2 74 179620 179639 842 533336 TCCTTTGAGGGATTGCCTCA Intron 2 66 179621 179640 843 533337 CTCCTTTGAGGGATTGCCTC Intron 2 76 179622 179641 844 533338 AACTTAGGACTTGGGACATT Intron 2 64 184575 184594 845 533339 TAACTTAGGACTTGGGACAT Intron 2 54 184576 184595 846 533340 CTAACTTAGGACTTGGGACA Intron 2 63 184577 184596 847 533341 CACTAACTTAGGACTTGGGA Intron 2 82 184579 184598 848 533342 TCACTAACTTAGGACTTGGG Intron 2 77 184580 184599 849 533343 GTCACTAACTTAGGACTTGG Intron 2 83 184581 184600 850 533344 TGGGCTAGATCAGGATTGGT Intron 2 81 188617 188636 851 533345 ATGGGCTAGATCAGGATTGG Intron 2 70 188618 188637 852 533346 CATGGGCTAGATCAGGATTG Intron 2 64 188619 188638 853 533347 ACCATGGGCTAGATCAGGAT Intron 2 82 188621 188640 854 533348 TACCATGGGCTAGATCAGGA Intron 2 88 188622 188641 855 533349 CTACCATGGGCTAGATCAGG Intron 2 87 188623 188642 856 533350 ATGAGCTTAGCAGTCACTTA Intron 2 83 189482 189501 857 533351 CATGAGCTTAGCAGTCACTT Intron 2 87 189483 189502 858 533352 CCATGAGCTTAGCAGTCACT Intron 2 92 189484 189503 859 533353 GTCTCAGCAAACCTGGGATA Intron 2 84 190283 190302 860 533354 TGTCTCAGCAAACCTGGGAT Intron 2 82 190284 190303 861 533355 ATGTCTCAGCAAACCTGGGA Intron 2 81 190285 190304 862 533356 GAATGTCTCAGCAAACCTGG Intron 2 76 190287 190306 863 533357 GGAATGTCTCAGCAAACCTG Intron 2 82 190288 190307 864 533358 AGGAATGTCTCAGCAAACCT Intron 2 85 190289 190308 865 533359 TACAGACATAGCTCTAACCT Intron 2 79 191139 191158 866 533360 ATACAGACATAGCTCTAACC Intron 2 79 191140 191159 867 533361 GATACAGACATAGCTCTAAC Intron 2 71 191141 191160 868 533362 TGGATACAGACATAGCTCTA Intron 2 79 191143 191162 869 533363 CTGGATACAGACATAGCTCT Intron 2 82 191144 191163 870 533364 GCTGGATACAGACATAGCTC Intron 2 95 191145 191164 871 533365 ACACTGTTTGTGAGGGTCAA Intron 2 87 191939 191958 872 533366 AACACTGTTTGTGAGGGTCA Intron 2 81 191940 191959 873 533367 CAACACTGTTTGTGAGGGTC Intron 2 85 191941 191960 874 533368 AACAACACTGTTTGTGAGGG Intron 2 65 191943 191962 875 533369 AAACAACACTGTTTGTGAGG Intron 2 76 191944 191963 876 533370 CAAACAACACTGTTTGTGAG Intron 2 67 191945 191964 877 533371 TTCAAGTTTAGGATCTGCAG Intron 2 73 196536 196555 878 533372 CTTCAAGTTTAGGATCTGCA Intron 2 88 196537 196556 879 533373 GCTTCAAGTTTAGGATCTGC Intron 2 86 196538 196557 880 533374 GGGCTTCAAGTTTAGGATCT Intron 2 67 196540 196559 881 533375 AGGGCTTCAAGTTTAGGATC Intron 2 66 196541 196560 882 533376 CAGGGCTTCAAGTTTAGGAT Intron 2 74 196542 196561 883 533377 TGTGGCTTTAATTCACTAAT Intron 2 84 198145 198164 884 533378 ATGTGGCTTTAATTCACTAA Intron 2 86 198146 198165 885 533379 TATGTGGCTTTAATTCACTA Intron 2 79 198147 198166 886 533380 GGTATGTGGCTTTAATTCAC Intron 2 83 198149 198168 887 533381 TGGTATGTGGCTTTAATTCA Intron 2 81 198150 198169 888 533382 GTGGTATGTGGCTTTAATTC Intron 2 86 198151 198170 889 533383 TCTGTGTTCAGTTGCATCAC Intron 2 75 199817 199836 890 533384 TTCTGTGTTCAGTTGCATCA Intron 2 82 199818 199837 891 533385 GTTCTGTGTTCAGTTGCATC Intron 2 86 199819 199838 892 533386 GTACTCATGAGGAGGCACTT Intron 2 81 201413 201432 893 533387 GGTACTCATGAGGAGGCACT Intron 2 82 201414 201433 894 533388 TGGTACTCATGAGGAGGCAC Intron 2 78 201415 201434 895 533389 ATTGGTACTCATGAGGAGGC Intron 2 64 201417 201436 896 533390 AATTGGTACTCATGAGGAGG Intron 2 47 201418 201437 897 533391 CAATTGGTACTCATGAGGAG Intron 2 54 201419 201438 898 533392 AAACTCTGCAACTCCAACCC Intron 2 69 205549 205568 899 533393 GAAACTCTGCAACTCCAACC Intron 2 64 205550 205569 900 533394 GGAAACTCTGCAACTCCAAC Intron 2 83 205551 205570 901 533395 ATGGAAACTCTGCAACTCCA Intron 2 88 205553 205572 902 533396 CATGGAAACTCTGCAACTCC Intron 2 70 205554 205573 903 533397 TCATGGAAACTCTGCAACTC Intron 2 69 205555 205574 904 533398 ACATCTGGATGTGAGGCTCG Intron 3 64 210559 210578 905 533399 CACATCTGGATGTGAGGCTC Intron 3 84 210560 210579 906 533400 GTCACATCTGGATGTGAGGC Intron 3 75 210562 210581 907 533401 TGTCACATCTGGATGTGAGG Intron 3 51 210563 210582 908 533402 CTGTCACATCTGGATGTGAG Intron 3 30 210564 210583 909

TABLE 135 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 NO: 2 SEQ ISIS Target % Start Stop ID NO Sequence region inhibition Site Site NO 523715 GTCAATTATGTGCTTTGCCT Intron 2 91 148907 148926 910 523716 ACATTCAAAATTCTTCCTTG Intron 2 50 149787 149806 911 523717 ATCCTGCATATATTTTATTG Intron 2 20 150588 150607 912 523718 CTGCTGGTGACTCTGCCTGA Intron 2 77 151388 151407 913 523719 AATGCTGAAGGATGGGCATC Intron 2 66 152204 152223 914 523720 TTATCCAGTAGTCAATATTA Intron 2 71 153004 153023 915 523721 TCTCATGTTAAAGTTCTTAA Intron 2 48 153831 153850 916 523722 TGCACTTGGACAACTGATAG Intron 2 29 154724 154743 917 523723 ACTCAACTTGAGGACAATAA Intron 2 88 155594 155613 918 523724 GACCAGGAAGAAAGGAACCT Intron 2 72 156394 156413 919 523725 TGCTACAATGCACAGGACAC Intron 2 80 157201 157220 920 523726 TCTGATATTTATTGCTGTAC Intron 2 73 158007 158026 921 523727 ATGCTTCCTTTAATAAATGT Intron 2 0 158807 158826 922 523728 AACATTTAGAACCTAGGAGA Intron 2 20 159610 159629 923 523729 CAAGCTTGCAAGTAGGAAAA Intron 2 51 160410 160429 924 523730 CCAGGCTGTTCATGCCAAGG Intron 2 26 161248 161267 925 523731 CCTGCCAAGGGCAAGCCAGG Intron 2 17 162064 162083 926 523732 TTTCACCTGGTGACTGGAAG Intron 2 51 163019 163038 927 523733 ATTTTCTACCATCAAAGAGA Intron 2 4 163943 163962 928 523734 GATTAAGTTTTCTTTAAAAA Intron 2 0 164746 164765 929 523735 CTTCCAGAAGAATTACCCAT Intron 2 56 165553 165572 930 523736 CAGTTTCTGCAGTATCCTAG Intron 2 77 166353 166372 931 523737 TATTTTGAAAATGAGATTCA Intron 2 0 167195 167214 932 523738 GTGGCCCGAGTAAAGATAAA Intron 2 21 167995 168014 933 523739 CCTGTCAATCCTCTTATATG Intron 2 37 168804 168823 934 523740 GGTGTTTCCATTTTCTTGAT Intron 2 65 169604 169623 935 523741 ACAGGGTCAAAAGTTCACTT Intron 2 44 170407 170426 936 523742 TAGGAAAGCTGAGAGAATCC Intron 2 35 171207 171226 937 523743 AGCATATGAAAAAATACTCA Intron 2 0 172101 172120 938 523744 CTTCAGAAATCAGCATCTGA Intron 2 45 172937 172956 939 523745 TTACAAGTGACAGTGTTTGT Intron 2 28 173737 173756 940 523746 ATCAGACCCTGAAGAATTTA Intron 2 29 174560 174579 941 523747 AGGAACCCATTTCATCCATT Intron 2 83 175372 175391 942 523748 CACATTGGTAACTTAAAGTT Intron 2 18 176263 176282 943 523749 TATTATCTGACTCATTTCTG Intron 2 16 177072 177091 944 523750 AAATAAGACAAAGAAAATTC Intron 2 0 177872 177891 945 523751 TTTTAAAAATAACCAATTCA Intron 2 0 178788 178807 946 523752 CTTTGAGGGATTGCCTCAGT Intron 2 66 179619 179638 947 523753 ACAGTCCTCATGAACAGATT Intron 2 37 180513 180532 948 523754 ACTATCATTAATAATATTGT Intron 2 0 181323 181342 949 523755 ATCTAGATTTGCCTTATAAG Intron 2 27 182123 182142 950 523756 TGGTTGAGGAAGACAGTCTC Intron 2 16 182962 182981 951 523757 TGGCTCATAACTTCCTTAGC Intron 2 43 183762 183781 952 523758 ACTAACTTAGGACTTGGGAC Intron 2 72 184578 184597 953 523759 CTTATAGCATTACTAAGTGG Intron 2 49 185403 185422 954 523760 TGGTGGCAGGAGAGAGGGAA Intron 2 48 186203 186222 955 523761 TTTGCCAGGAAATCTTGAAA Intron 2 35 187003 187022 956 523762 ATAACTTTTCTCTGAAATTT Intron 2 8 187803 187822 957 523763 CCATGGGCTAGATCAGGATT Intron 2 59 188620 188639 958 523764 TGAGCTTAGCAGTCACTTAG Intron 2 62 189481 189500 959 523765 AATGTCTCAGCAAACCTGGG Intron 2 62 190286 190305 960 523766 GGATACAGACATAGCTCTAA Intron 2 75 191142 191161 961 523767 ACAACACTGTTTGTGAGGGT Intron 2 66 191942 191961 962 523768 TCTATTTTCTAATAGCTGTT Intron 2 49 192742 192761 963 523769 GGCCCCACCTCTGACCTTCA Intron 2 7 193542 193561 964 523770 TGGTAAAGCTAGAAAAAAAA Intron 2 0 194346 194365 965 523771 AAGTGGTAAATATGATCACA Intron 2 23 195159 195178 966 523772 GGCTTCAAGTTTAGGATCTG Intron 2 52 196539 196558 967 523773 TTGTTGACACTCTCTTTTGG Intron 2 18 197348 197367 968 523774 GTATGTGGCTTTAATTCACT Intron 2 71 198148 198167 969 523775 AATTAGTTGTTTTGGCAAAT Intron 2 14 198988 199007 970 523776 CTGTGTTCAGTTGCATCACG Intron 2 75 199816 199835 971 523777 AATGTGGAAGTTTCCTAACA Intron 2 15 200616 200635 972 523778 TTGGTACTCATGAGGAGGCA Intron 2 58 201416 201435 973 523779 TTTCTCTGTGTTTAAAATTG Intron 2 13 202308 202327 974 523780 GTAAAGCACAATGAACAAAA Intron 2 21 203115 203134 975 523781 ATCACAGATCTTTGCTACAA Intron 2 51 203915 203934 976 523782 TCCTGCCTTTCTGAACCAAA Intron 2 50 204721 204740 977 523783 TGGAAACTCTGCAACTCCAA Intron 2 58 205552 205571 978 523784 ACACAGTAGGGAACAATTTT Intron 2 8 206412 206431 979 523785 AGACAGATGGTGAAATGATG Intron 2 0 207219 207238 980 523786 AAACAGAAAGAGAAGAAAAC Intron 2 0 208117 208136 981 523787 CTTAGATAAATACTTCAAGA Intron 3 0 208938 208957 982 523788 AGCCACTTCTTTTACAACCT Intron 3 0 209742 209761 983 523789 TCACATCTGGATGTGAGGCT Intron 3 80 210561 210580 984 523790 GACTGAAACTTAAAGGTGGG Intron 3 7 211399 211418 985 523791 AAAGATGTGCAATCATCTAA Intron 3 44 212204 212223 986

TABLE 136 Inhibition of GHR mRNA by 3-10-4 MOE gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ ID NO: ID NO: SEQ ISIS Target % 2 Start 2 Stop ID NO Sequence region inhibition Site Site NO 539360 GCTGGTGACTCTGCCTG Intron 2 95 151389 151405 987 539361 TGCTGGTGACTCTGCCT Intron 2 95 151390 151406 988 539362 CTGCTGGTGACTCTGCC Intron 2 93 151391 151407 989 539363 AGTAGTCAATATTATTT Intron 2 31 153001 153017 990 539364 CAGTAGTCAATATTATT Intron 2 13 153002 153018 991 539365 CCAGTAGTCAATATTAT Intron 2 34 153003 153019 992 539366 CCTTTGGGTGAATAGCA Intron 2 64 153921 153937 993 539367 ACCTTTGGGTGAATAGC Intron 2 78 153922 153938 994 539368 CACCTTTGGGTGAATAG Intron 2 40 153923 153939 995 539369 CAACTTGAGGACAATAA Intron 2 38 155594 155610 996 539370 TCAACTTGAGGACAATA Intron 2 63 155595 155611 997 539371 CTCAACTTGAGGACAAT Intron 2 81 155596 155612 998 539372 CAGGAAGAAAGGAACCT Intron 2 70 156394 156410 999 539373 CCAGGAAGAAAGGAACC Intron 2 59 156395 156411 1000 539374 ACCAGGAAGAAAGGAAC Intron 2 43 156396 156412 1001 539375 TGCAGTCATGTACACAA Intron 2 93 156594 156610 1002 539376 CTGCAGTCATGTACACA Intron 2 91 156595 156611 1003 539377 TCTGCAGTCATGTACAC Intron 2 87 156596 156612 1004 539378 TGGTTTGTCAATCCTTT Intron 2 95 156889 156905 1005 539379 TTGGTTTGTCAATCCTT Intron 2 97 156890 156906 1006 539380 CTTGGTTTGTCAATCCT Intron 2 97 156891 156907 1007 539381 TACAATGCACAGGACAC Intron 2 65 157201 157217 1008 539382 CTACAATGCACAGGACA Intron 2 85 157202 157218 1009 539383 GCTACAATGCACAGGAC Intron 2 96 157203 157219 1010 539384 GATATTTATTGCTGTAC Intron 2 43 158007 158023 1011 539385 TGATATTTATTGCTGTA Intron 2 35 158008 158024 1012 539386 CTGATATTTATTGCTGT Intron 2 38 158009 158025 1013 539387 AGGGTCTTTACAAAGTT Intron 2 61 162751 162767 1014 539388 CAGGGTCTTTACAAAGT Intron 2 65 162752 162768 1015 539389 CCAGGGTCTTTACAAAG Intron 2 88 162753 162769 1016 539390 TTCTGCAGTATCCTAGC Intron 2 72 166352 166368 1017 539391 TTTCTGCAGTATCCTAG Intron 2 53 166353 166369 1018 539392 GTTTCTGCAGTATCCTA Intron 2 84 166354 166370 1019 539393 AGTTTCTGCAGTATCCT Intron 2 78 166355 166371 1020 539394 CAGTTTCTGCAGTATCC Intron 2 77 166356 166372 1021 539395 CAAATTCCAGTCCTAGG Intron 2 60 172746 172762 1022 539396 CCAAATTCCAGTCCTAG Intron 2 75 172747 172763 1023 539397 TCCAAATTCCAGTCCTA Intron 2 62 172748 172764 1024 539398 AACCCATTTCATCCATT Intron 2 82 175372 175388 1025 539399 GAACCCATTTCATCCAT Intron 2 86 175373 175389 1026 539400 GGAACCCATTTCATCCA Intron 2 84 175374 175390 1027 539401 GCTTCATGTCTTTCTAG Intron 2 88 189119 189135 1028 539402 TGCTTCATGTCTTTCTA Intron 2 77 189120 189136 1029 539403 GTGCTTCATGTCTTTCT Intron 2 95 189121 189137 1030 539404 TGAGCTTAGCAGTCACT Intron 2 92 189484 189500 1031 539405 CATGAGCTTAGCAGTCA Intron 2 82 189486 189502 1032 539406 TACAGACATAGCTCTAA Intron 2 45 191142 191158 1033 539407 ATACAGACATAGCTCTA Intron 2 53 191143 191159 1034 539408 GATACAGACATAGCTCT Intron 2 67 191144 191160 1035 539409 TGTGGCTTTAATTCACT Intron 2 70 198148 198164 1036 539410 ATGTGGCTTTAATTCAC Intron 2 40 198149 198165 1037 539411 TATGTGGCTTTAATTCA Intron 2 35 198150 198166 1038 539412 TGTTCAGTTGCATCACG Intron 2 84 199816 199832 1039 539413 GTGTTCAGTTGCATCAC Intron 2 80 199817 199833 1040 539414 TGTGTTCAGTTGCATCA Intron 2 74 199818 199834 1041 539415 CATCTGGATGTGAGGCT Intron 3 82 210561 210577 1042 539416 ACATCTGGATGTGAGGC Intron 3 86 210562 210578 1043 539417 CACATCTGGATGTGAGG Intron 3 55 210563 210579 1044 539418 TCAGGTAATTTCTGGAA Intron 3 35 219019 219035 1045 539419 CTCAGGTAATTTCTGGA Intron 3 44 219020 219036 1046 539420 TCTCAGGTAATTTCTGG Intron 3 31 219021 219037 1047 539421 TTGCTTATTTACCTGGG Intron 3 0 225568 225584 1048 539422 TTTGCTTATTTACCTGG Intron 3 38 225569 225585 1049 539423 TTTTGCTTATTTACCTG Intron 3 33 225570 225586 1050 539424 ATGATGTTACTACTACT Intron 3 29 229618 229634 1051 539425 AATGATGTTACTACTAC Intron 3 10 229619 229635 1052 539426 CAATGATGTTACTACTA Intron 3 0 229620 229636 1053 539427 CCCCTAGAGCAATGGTC Intron 3 67 232826 232842 1054 539428 CCCCCTAGAGCAATGGT Intron 3 65 232827 232843 1055 539429 TCCCCCTAGAGCAATGG Intron 3 45 232828 232844 1056 539430 TCAATTGCAGATGCTCT Intron 3 78 237675 237691 1057 539431 CTCAATTGCAGATGCTC Intron 3 82 237676 237692 1058 539432 GCTCAATTGCAGATGCT Intron 3 92 237677 237693 1059 539433 AGCTCAATTGCAGATGC Intron 3 85 237678 237694 1060 539434 GTATATTCAGTCCAAGG Intron 3 73 248231 248247 1061 539435 AGTATATTCAGTCCAAG Intron 3 70 248232 248248 1062 539436 CAGTATATTCAGTCCAA Intron 3 40 248233 248249 1063

TABLE 137 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 1 and 3 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 ISIS Target % Start Stop SEQ ID NO Sequence region inhibition Site Site NO 532502 GAGTATTTCAGGCTGGAAAA Intron 3 43 214623 214642 1064 533404 GTAACTCAGGAATGGAAAAC Intron 1 56 26501 26520 1065 113035 113054 121992 122011 533405 AGTAACTCAGGAATGGAAAA Intron 1 41 26502 26521 1066 113036 113055 121993 122012 533406 AAGTAACTCAGGAATGGAAA Intron 1 43 26503 26522 1067 113037 113056 121994 122013 533407 GAGATTTCAAATAAATCTCA Intron 1 0 143207 143226 1068 143235 143254 143263 143282 143291 143310 143319 143338 143347 143366 143375 143394 143403 143422 143431 143450 143459 143478 533408 TGAGATTTCAAATAAATCTC Intron 1 11 143208 143227 1069 143236 143255 143264 143283 143292 143311 143320 143339 143348 143367 143376 143395 143404 143423 143432 143451 143460 143479 533409 GTGAGATTTCAAATAAATCT Intron 1 0 143209 143228 1070 143237 143256 143265 143284 143293 143312 143321 143340 143349 143368 143377 143396 143405 143424 143433 143452 143461 143480 533410 TGTGAGATTTCAAATAAATC Intron 1 0 143210 143229 1071 143238 143257 143266 143285 143294 143313 143322 143341 143350 143369 143378 143397 143406 143425 143434 143453 143462 143481 533411 TTGTGAGATTTCAAATAAAT Intron 1 10 143183 143202 1072 143211 143230 143239 143258 143267 143286 143295 143314 143323 143342 143351 143370 143379 143398 143407 143426 143435 143454 143463 143482 533412 TTTGTGAGATTTCAAATAAA Intron 1 0 143184 143203 1073 143212 143231 143240 143259 143296 143315 143324 143343 143352 143371 143380 143399 143464 143483 533413 CTTTGTGAGATTTCAAATAA Intron 1 20 143185 143204 1074 143213 143232 143241 143260 143297 143316 143325 143344 143353 143372 143381 143400 143465 143484 533414 ACTTTGTGAGATTTCAAATA Intron 1 57 143186 143205 1075 143214 143233 143242 143261 143298 143317 143326 143345 143354 143373 143382 143401 143466 143485 533415 CACTTTGTGAGATTTCAAAT Intron 1 69 143187 143206 1076 143215 143234 143243 143262 143299 143318 143327 143346 143355 143374 143383 143402 143467 143486 533895 AGTATTTCAGGCTGGAAAAA Intron 3 35 214622 214641 1077 533896 TGAGTATTTCAGGCTGGAAA Intron 3 55 214624 214643 1078 533897 TCTGAGTATTTCAGGCTGGA Intron 3 71 214626 214645 1079 533898 ATCTGAGTATTTCAGGCTGG Intron 3 77 214627 214646 1080 533899 TATCTGAGTATTTCAGGCTG Intron 3 58 214628 214647 1081 533900 TTTTGTGTTATGCCTTGAGG Intron 3 51 221483 221502 1082 533901 TTTTTGTGTTATGCCTTGAG Intron 3 55 221484 221503 1083 533902 ATTTTTGTGTTATGCCTTGA Intron 3 57 221485 221504 1084 533903 ATATTTTTGTGTTATGCCTT Intron 3 56 221487 221506 1085 533904 AATATTTTTGTGTTATGCCT Intron 3 61 221488 221507 1086 533905 AAATATTTTTGTGTTATGCC Intron 3 18 221489 221508 1087 533906 TTGCTTATTTACCTGGGTAA Intron 3 58 225565 225584 1088 533907 TTTGCTTATTTACCTGGGTA Intron 3 64 225566 225585 1089 533908 TTTTGCTTATTTACCTGGGT Intron 3 77 225567 225586 1090 533909 CCTTTTGCTTATTTACCTGG Intron 3 69 225569 225588 1091 533910 GCCTTTTGCTTATTTACCTG Intron 3 69 225570 225589 1092 533911 TGCCTTTTGCTTATTTACCT Intron 3 55 225571 225590 1093 533912 ATGATGTTACTACTACTCAA Intron 3 60 229615 229634 1094 533913 AATGATGTTACTACTACTCA Intron 3 48 229616 229635 1095 533914 CAATGATGTTACTACTACTC Intron 3 57 229617 229636 1096 533915 TCCAATGATGTTACTACTAC Intron 3 69 229619 229638 1097 533916 TTCCAATGATGTTACTACTA Intron 3 74 229620 229639 1098 533917 ATTCCAATGATGTTACTACT Intron 3 74 229621 229640 1099 533918 CCCCTAGAGCAATGGTCTAG Intron 3 71 232823 232842 1100 533919 CCCCCTAGAGCAATGGTCTA Intron 3 44 232824 232843 1101 533920 TCCCCCTAGAGCAATGGTCT Intron 3 54 232825 232844 1102 533921 TATCCCCCTAGAGCAATGGT Intron 3 62 232827 232846 1103 533922 ATATCCCCCTAGAGCAATGG Intron 3 50 232828 232847 1104 533923 AATATCCCCCTAGAGCAATG Intron 3 61 232829 232848 1105 533924 GCTCACATTTGGAAGACAGT Intron 3 68 233623 233642 1106 533925 GGCTCACATTTGGAAGACAG Intron 3 74 233624 233643 1107 533926 AGGCTCACATTTGGAAGACA Intron 3 56 233625 233644 1108 533927 AGAGGCTCACATTTGGAAGA Intron 3 34 233627 233646 1109 533928 TAGAGGCTCACATTTGGAAG Intron 3 18 233628 233647 1110 533929 TTAGAGGCTCACATTTGGAA Intron 3 19 233629 233648 1111 533930 CTCAATTGCAGATGCTCTGA Intron 3 66 237673 237692 1112 533931 GCTCAATTGCAGATGCTCTG Intron 3 72 237674 237693 1113 533932 AGCTCAATTGCAGATGCTCT Intron 3 74 237675 237694 1114 533933 AAAGCTCAATTGCAGATGCT Intron 3 66 237677 237696 1115 533934 TAAAGCTCAATTGCAGATGC Intron 3 59 237678 237697 1116 533935 ATAAAGCTCAATTGCAGATG Intron 3 23 237679 237698 1117 533936 GTGAGTCCATTAAACCTCTT Intron 3 73 244873 244892 1118 533937 TGTGAGTCCATTAAACCTCT Intron 3 73 244874 244893 1119 533938 ACTGTGAGTCCATTAAACCT Intron 3 17 244876 244895 1120 533939 AACTGTGAGTCCATTAAACC Intron 3 19 244877 244896 1121 533940 GAACTGTGAGTCCATTAAAC Intron 3 28 244878 244897 1122 533941 ATATTGAAAGGCCCATCAAA Intron 3 13 246498 246517 1123 533942 AATATTGAAAGGCCCATCAA Intron 3 31 246499 246518 1124 533943 AAATATTGAAAGGCCCATCA Intron 3 51 246500 246519 1125 533944 GAAAATATTGAAAGGCCCAT Intron 3 22 246502 246521 1126 533945 GGAAAATATTGAAAGGCCCA Intron 3 42 246503 246522 1127 533946 AGGAAAATATTGAAAGGCCC Intron 3 28 246504 246523 1128 533947 GTATATTCAGTCCAAGGATC Intron 3 65 248228 248247 1129 533948 AGTATATTCAGTCCAAGGAT Intron 3 63 248229 248248 1130 533949 CAGTATATTCAGTCCAAGGA Intron 3 67 248230 248249 1131 533950 AACAGTATATTCAGTCCAAG Intron 3 56 248232 248251 1132 533951 AAACAGTATATTCAGTCCAA Intron 3 60 248233 248252 1133 533952 AAAACAGTATATTCAGTCCA Intron 3 59 248234 248253 1134 533953 TCTATTGTTGCCACCTTTAT Intron 3 45 252838 252857 1135 533954 TTCTATTGTTGCCACCTTTA Intron 3 52 252839 252858 1136 533955 TTTCTATTGTTGCCACCTTT Intron 3 46 252840 252859 1137 533956 AGTTTCTATTGTTGCCACCT Intron 3 59 252842 252861 1138 533957 CAGTTTCTATTGTTGCCACC Intron 3 41 252843 252862 1139 533958 CCAGTTTCTATTGTTGCCAC Intron 3 48 252844 252863 1140

TABLE 138 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 3 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 SEQ ISIS % Start Stop ID NO Sequence inhibition Site Site NO 532454 GCAGAACTGATTGCTTACTT 78 182862 182881 1141 532455 AGGTCATAAGATTTTCATTT 48 183533 183552 1142 532456 GCCTCTGGCCATAAAGAAAT 54 183578 183597 1143 532457 AAAGTTTAAGAGGCACCCCA 31 184508 184527 1144 532458 GAATAAGCACAAAAGTTTAA 28 184519 184538 1145 532459 GAACCAAATAAACCTCTCTT 52 185452 185471 1146 532460 ATGTTGAAATTTGATCCCCA 79 185763 185782 1147 532461 TGTGAGAGCTCACTCACTAT 42 186134 186153 1148 532462 CTTGTGAGAGCTCACTCACT 72 186136 186155 1149 532463 ACATGGTGGCAGGAGAGAGG 42 186206 186225 1150 532464 CTAGAAAGAAACTACCTGAG 12 186341 186360 1151 532465 AACTTCAGTTGTAAAATAAT 27 187044 187063 1152 532466 GAAAAGGATTTTGAGATTTC 43 188897 188916 1153 532467 CTTAGCTGTCAAGGCCCTTT 80 189084 189103 1154 532468 TGTGCTTCATGTCTTTCTAG 88 189119 189138 1155 532469 CCCTTGAACATGCTATCCTT 85 189256 189275 1156 532470 CTTGCAGGGATGCATCTCAG 87 189625 189644 1157 532471 TCTCTTGCACATCTAATTTC 82 189656 189675 1158 532472 CTTCCAGCACAACCCATCAC 77 190109 190128 1159 532473 GTAACTACATTCCCTTTATC 52 190860 190879 1160 532474 AGTAACTACATTCCCTTTAT 58 190861 190880 1161 532475 CAGATAGCACAGGGCTAAAA 84 190979 190998 1162 532476 AGAATCAGGAATGTTTGCCT 86 192904 192923 1163 532477 TGACTCAATCATTTAGACTT 45 192990 193009 1164 532478 TCAACAGTCAATGGACTTGT 71 193042 193061 1165 532479 AATTTCTACTGCTATGATGC 75 194806 194825 1166 532480 ATGGTTCCAAATTTCTATCT 86 195704 195723 1167 532481 CTGTATGGCTTTAAGTATTC 63 196756 196775 1168 532482 AACTTATGAACTGTTCACCA 86 198307 198326 1169 532483 AATAAGCTTGAAGTCTGAAG 63 199520 199539 1170 532484 TAGTTATCTAACTGCCCAAT 77 199544 199563 1171 532485 TTCTGCAAAGCTTCCCAGTA 72 200314 200333 1172 532486 ACAACTTCAAGCTTCACATA 65 200599 200618 1173 532487 GAATCAATGTTCTGGCAAGA 52 201842 201861 1174 532488 CAGCCTTTCAGCTGTGAAAG 52 204181 204200 1175 532489 AACAATGCCAAGAAATCTAT 74 204369 204388 1176 532490 CCCACAGTAACAATGCCAAG 90 204377 204396 1177 532491 TTTTACCTCCCAGTGAAACT 34 205896 205915 1178 532492 TAATTGTTGATCCATGATGT 5 208856 208875 1179 532493 GTTGGAGAGACAAGTTTAAC 29 208975 208994 1180 532494 AGTCATAAAATTCAAATTAT 39 209537 209556 1181 532495 GGCCTTGGGCACACTTTCTC 82 207510 207529 1182 210189 210208 532496 AAGTTTTTATTGAAGTTAAT 0 212551 212570 1183 532497 AAGAAAAATTAGGAAGCTAG 31 212649 212668 1184 532498 CAGGGAGATAAGTTTATTCA 61 212797 212816 1185 532499 ATTTAATACACATTGGAATA 15 213390 213409 1186 532500 GTAGGACTATTTATGATTCC 86 213914 213933 1187 532501 CACTCTCTTGGGCTGTTAAG 82 214479 214498 1188 532502 GAGTATTTCAGGCTGGAAAA 66 214623 214642 1064 532503 TTGTTTGAGTTCCAAAAGAA 39 214932 214951 1189 532504 TTTGCCATGAGACACACAAT 77 215932 215951 1190 532505 CACCAAACCTCAGAGACATG 80 216468 216487 1191 532506 CCACTGTTAAGTGATGCATG 83 217480 217499 1192 532507 CTCTCAGGTAATTTCTGGAA 86 219019 219038 1193 532508 GCTCCTCACAATGACCCTTT 84 219452 219471 1194 532509 GGGACTGGCACTGGTAATTT 56 220062 220081 1195 532510 CTAACCATTAGTTACTGTAT 69 220558 220577 1196 532511 GGATTTTAGGTTCTTGCTGT 51 221588 221607 1197 532512 TGAATCATATACTGATATCA 63 222914 222933 1198 532513 TTGAGGTATTAAATTTTAAA 0 223001 223020 1199 532514 AGTTTGTAATGTAGTGATTT 19 223156 223175 1200 532515 AAATATTTGATAGCTCACAT 18 224409 224428 1201 532516 AGAAATATTTGATAGCTCAC 57 224411 224430 1202 532517 CCACATTTCAAATGTTCTCT 80 224717 224736 1203 532518 GCAGGAAGAGTGGCATGGAC 59 224750 224769 1204 532519 CACTTATCCAAATGCAGAGA 82 225742 225761 1205 532520 CAAGGTAATGGGAGGCTAGC 47 225903 225922 1206 532521 ATAGTCAAAGCTAAGGATAT 4 226177 226196 1207 532522 GTAATTTCATTCATGCTTCC 67 226804 226823 1208 532523 GTCCACATTCAGCTGTGTGT 72 231912 231931 1209 532524 TCATTCAGGAAATTCTGCTA 62 232286 232305 1210 532525 AACATGTCTCATTCAGGAAA 71 232294 232313 1211 532526 TAACATGTCTCATTCAGGAA 85 232295 232314 1212 532527 AGATTCCTCAAATTCAGTGA 66 232389 232408 1213 532528 TAAGCGGAAAAGGAGAAAAG 0 233684 233703 1214 532529 AAAGCAAGAGAATTCCTAAA 32 234203 234222 1215 532530 AATGAACCTTTAACTTAGTA 40 234876 234895 1216

TABLE 139 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 3-8 and intron-exonic regions of SEQ ID NO: 2 SEQ SEQ ID NO: ID NO: SEQ ISIS % 2 Start 2 Stop ID NO Sequence Target region inhibition Site Site NO 523792 AAAGCTTTGTGGATAAAGTT Intron 3 44 213025 213044 1217 523793 GAAGGAAAGGTTCTGTGGAA Intron 3 38 213825 213844 1218 523794 CTGAGTATTTCAGGCTGGAA Intron 3 84 214625 214644 1219 523795 TTGAATTATCCCTTTAAAAA Intron 3 38 215446 215465 1220 523796 TTTAGAATGGTTTGGCATAC Intron 3 66 216365 216384 1221 523797 GATATGTCCACATTGATTAG Intron 3 65 218132 218151 1222 523798 ATTATTTAAGCTTCTACTTT Intron 3 44 218973 218992 1223 523799 ATACATGGCAATTAAAAGAT Intron 3 26 219886 219905 1224 523800 TGAGATAGTGTGGGAAATAT Intron 3 18 220686 220705 1225 523801 TATTTTTGTGTTATGCCTTG Intron 3 73 221486 221505 1226 523802 TTATTAACTAGAATATGCCT Intron 3 16 223110 223129 1227 523803 GATTATTCTATTTTTATTTT Intron 3 33 223948 223967 1228 523804 AGGAAGAGTGGCATGGACAT Intron 3 43 224748 224767 1229 523805 CTTTTGCTTATTTACCTGGG Intron 3 84 225568 225587 1230 523806 TTTATATTATTAATATCATT Intron 3 31 226371 226390 1231 523807 GGTACATGGCTTTTAAGTGG Intron 3 53 227218 227237 1232 523808 AATATTGGTCAGGTTTAAGA Intron 3 28 228018 228037 1233 523809 ATTTCATCTCTTTCTTAGTT Intron 3 45 228818 228837 1234 523810 CCAATGATGTTACTACTACT Intron 3 89 229618 229637 1235 523811 GTTCCCCCAACCCCTTGGAA Intron 3 28 230418 230437 1236 523812 TATAGGAAGTGAGATGTATG Intron 3 46 231218 231237 1237 523813 ATTATTCTAGAAGAAGATTT Intron 3 12 232018 232037 1238 523814 ATCCCCCTAGAGCAATGGTC Intron 3 79 232826 232845 1239 523815 GAGGCTCACATTTGGAAGAC Intron 3 69 233626 233645 1240 523816 TACACAAATCCAAGGCAGAG Intron 3 57 234447 234466 1241 523817 AGGAAGAGTGGGAGTGTTAC Intron 3 35 235258 235277 1242 523818 GTCCCTGACTAGGCATTTTG Intron 3 43 236071 236090 1243 523819 AAGCTCAATTGCAGATGCTC Intron 3 80 237676 237695 1244 523820 CTGTGAGTCCATTAAACCTC Intron 3 81 244875 244894 1245 523821 TGAAATGTGGCTAGTGTGAC Intron 3 51 245701 245720 1246 523822 AAAATATTGAAAGGCCCATC Intron 3 68 246501 246520 1247 523823 AATGTCAATAGTGCCCTATT Intron 3 48 247431 247450 1248 523824 ACAGTATATTCAGTCCAAGG Intron 3 82 248231 248250 1249 523825 TGTCTATTTAAGTTTGTTGC Intron 3 45 250001 250020 1250 523826 TTCAAGTACTGTCATGAATA Intron 3 47 251214 251233 1251 523827 TTTCTTTTTCTTAAACTAAG Intron 3 11 252041 252060 1252 523828 GTTTCTATTGTTGCCACCTT Intron 3 70 252841 252860 1253 523829 AAGGCCACATATTATAGTAT Intron 3 29 253698 253717 1254 523830 ACCTGAACTATTAATTTCTT Intron 3 19 255397 255416 1255 523831 GAATGGGCTGAGTAGTTGAA Intron 3 47 256197 256216 1256 523832 TGATGAACATTGCTAATTTG Intron 3 26 257018 257037 1257 523833 ATCTTGCCTCGATGAAAGTT Intron 3 17 257818 257837 1258 523834 TTAAGTGGCACAGCCATGAT Intron 3 9 258774 258793 1259 523835 AATGAGTTAAGTTGGAACAC Intron 3 25 261294 261313 1260 523836 TCCTTAGTAGAATGCCTGGA Intron 3 57 263338 263357 1261 523837 TATGTAGAAAAATAAGCTGG Intron 3 0 266514 266533 1262 523838 GCCGAGGCAGGCACCTGAGT Intron 3 43 267375 267394 1263 523839 TGGTACCTATATTGAGAGGT Intron 4 46 269052 269071 1264 523840 TTAAGGAAAAATATAGTATA Intron 4 7 269854 269873 1265 523841 TTATTTATGTGTCAGGGATG Intron 4 28 270668 270687 1266 523842 CAAAAGTTAAGTGCTTTAGG Intron 4 10 271468 271487 1267 523843 TTCATAGATGTCTAAGGAAT Intron 4 32 273341 273360 1268 523844 ACCTGTGATTTACCTATTTC Exon 5-intron 5 18 274185 274204 1269 junction 523845 TGCCTAGAAAACCACATAAA Intron 5 38 274985 275004 1270 523846 AAACATCCTCAAAGGTACCT Intron 5 64 275808 275827 1271 523847 CTTCCCTGAGACACACACAT Intron 5 35 276617 276636 1272 523848 CTTCTTCAATCTTCTCATAC Intron 5 33 278288 278307 1273 523849 TACCATTTTCCATTTAGTTT Exon 6-intron 6 7 279088 279107 1274 junction 523850 ATTGGCATCTTTTTCAGTGG Intron 6 34 279902 279921 1275 523851 TCAAGCTCACGGTTGGAGAC Intron 6 36 280799 280818 1276 523852 AAATGAAATCAGTATGTTGA Intron 6 0 281622 281641 1277 523853 TGATTTATCACAAAGGTGCT Intron 6 29 282437 282456 1278 523854 AAAACAGTAGAAAAGATTAA Intron 6 14 284073 284092 1279 523855 CTACATCACAGCAGTCAGAA Intron 6 23 285187 285206 1280 523856 AAAAGATGTAAGTGTGACAT Intron 6 28 286349 286368 1281 286919 286938 523857 TTACAAGAACTGCTAAAGGG Intron 6 15 287151 287170 1282 523858 ATAAAGAAAAAGTTAACTGA Intron 6 9 287982 288001 1283 523859 AGATAATATACTTCTTCTAT Intron 6 4 288809 288828 1284 523860 CCTTCTTCACATGTAAATTG Exon 7-intron 7 19 290456 290475 1285 junction 523861 TTTCTATGTAGCTTGTGGTT Intron 7 30 291258 291277 1286 523862 AGGCAGAGTTTTTATTGATA Intron 7 19 292058 292077 1287 523863 ATAGTCACCAGCCTAAGCCT Intron 8 28 292858 292877 1288 523864 AGACTTTTAGCATGCTTGAC Intron 8 56 293658 293677 1289 523865 TTTACAGCCCTACAGTTCTA Intron 8 7 294464 294483 1290 523866 CCAGAGAACCTGACTCCAAA Intron 8 6 295330 295349 1291 523867 CAGAAGAAAATATTAGACAG Intron 8 10 296993 297012 1292

TABLE 140 Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting introns 3-8 of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 SEQ ISIS Target % Start Stop ID NO Sequence Region inhibition Site Site NO 532531 TATTATACTTCTAAATTCCC Intron 3 70 236716 236735 1293 532532 TAAAAGCAAGAAAAAGGAAC Intron 3 52 236889 236908 1294 532533 CCTAATTTATATGAACAAAC Intron 3 56 237177 237196 1295 532534 TGCAATGCCTTAGCCTAAAA Intron 3 86 238087 238106 1296 532535 CACCACCATTATTACACTAC Intron 3 75 238186 238205 1297 532536 AAATAAATCAGATTATTATA Intron 3 52 238242 238261 1298 532537 CTTAGATCTGTGCTGTCCAA Intron 3 81 245758 245777 1299 532538 GTTAGTGTTAGATTCTTTGA Intron 3 67 246152 246171 1300 532539 CATGCTCACGGCTGTGTTAC Intron 3 66 246248 246267 1301 532540 CCCATCAAATACTGAGTTCT Intron 3 86 246487 246506 1302 532541 GAAAGTAGTGATTAATGAGA Intron 3 38 247012 247031 1303 532542 ATTAATCAACAAGTGGCATT Intron 3 72 247203 247222 1304 532543 TTTAATTTTAGGGTTTAGAG Intron 3 48 248344 248363 1305 532544 CTTGCTACCACTAGAGCCTT Intron 3 69 248694 248713 1306 532545 ACCACTGACTTATATCATTT Intron 3 58 248743 248762 1307 532546 TTCCCCATTGCTAATTTTGT Intron 3 48 251601 251620 1308 532547 TCCTGAAACTTAGTAGCTGG Intron 3 83 253147 253166 1309 532548 TGTCTTAAAAAGGAATAAAA Intron 3 52 253785 253804 1310 532549 CCTATAATAAAGTATTGTCT Intron 3 70 253800 253819 1311 532550 ATGTAAAATGGTATAGCTAC Intron 3 50 254040 254059 1312 532551 AACCCTCACACACTTCTGTT Intron 3 71 254064 254083 1313 532552 ATTCTGCATAAGCAGTGTTT Intron 3 53 254246 254265 1314 532553 TTACTACCCTGAAGAAGAAC Intron 3 35 254314 254333 1315 532554 AAGACCTATAACTTACTACC Intron 3 49 254326 254345 1316 532555 TTTCACAAGATTTACTTGGT Intron 3 77 254641 254660 1317 532556 CAGTTGTGATTGTCAACCTA Intron 3 77 257073 257092 1318 532557 AATCTTGCCTCGATGAAAGT Intron 3 57 257819 257838 1319 532558 TGGCCTAAATGTATCAGTTA Intron 3 66 259157 259176 1320 532559 AGGCTTTGGGTAAAATCTTT Intron 3 67 259184 259203 1321 532560 TATGATTTTTAAAGATTAAA Intron 3 20 261419 261438 1322 532561 GTACAGTGAAAAAGATGTGT Intron 3 56 263666 263685 1323 532562 GACAGGTATGAAGCAAAACA Intron 3 64 267033 267052 1324 532563 TGAGCTGAGGGTCTTTGCCG Intron 3 61 267391 267410 1325 532564 AGGCTGAGTTGTACACAAAC Intron 4 52 269422 269441 1326 532565 ATGAGGAGGCTGAGTTGTAC Intron 4 43 269428 269447 1327 532566 TCATAAAGTGGGCCCAGCTT Intron 4 70 270044 270063 1328 532567 ACTCCTAATCCCTCAGTTTT Intron 4 62 270492 270511 1329 532568 TTTACATGCAAGGAGCTGAG Intron 4 61 271047 271066 1330 532569 TAATGCCCTTTCTCCCTACT Intron 4 60 271215 271234 1331 532570 CCTGTTTAGATTATCCCAAA Intron 4 62 271763 271782 1332 532571 CATGATTCACAGAATTTCTC Intron 4 56 271831 271850 1333 532572 AGTTAGAAAACTCAAAGTAT Intron 4 2 271915 271934 1334 532573 TCAAATGTACTTAGCATAAG Intron 4 9 271947 271966 1335 532574 ATATCAAATGTACTTAGCAT Intron 4 59 271950 271969 1336 532575 AAAGTTCAGAAGAGGGAATG Intron 4 51 273233 273252 1337 532576 AATTCCCATCTGAGTAGTTT Intron 4 56 273440 273459 1338 532577 GTCCCCTAATTTCAGGCTAA Intron 4 31 273471 273490 1339 532578 CTATGTCAAATGAAACAAAA Intron 5 38 274205 274224 1340 532579 TGATTATGCTTTGTGATAAA Intron 5 42 274624 274643 1341 532580 TCCAGCTGACTAGGAGGGCT Intron 5 7 275732 275751 1342 532581 CATACCAGTCTCCTCGCTCA Intron 5 0 276738 276757 1343 532582 ATATAACAGAATCCAACCAT Intron 5 47 277045 277064 1344 278361 278380 532583 TGCAAAATGGCCAAACTACA Intron 5 56 277577 277596 1345 532584 TCTTCCTAGCCACATGTGAT Intron 5 32 278227 278246 1346 532585 TACCATGCTCTCTAATTGCC Intron 6 47 279624 279643 1347 532586 AGTGATCTGTGCCAGGCTGC Intron 6 65 279848 279867 1348 532587 AAGTTACAGAACAGATATCT Intron 6 61 280012 280031 1349 532588 GTATTGTGAAAATAGTACTG Intron 6 45 280226 280245 1350 532589 AAACACTATCAAGCTCACGG Intron 6 54 280807 280826 1351 532590 TTCAAGAAAAGTCTTCAAAT Intron 6 24 280831 280850 1352 532591 GGATCATTTCCCCATGCATG Intron 6 52 280982 281001 1353 532592 ATATTATATTAAGAAAAATG Intron 6 4 281422 281441 1354 532593 CTCCCATGTTCATTACTTAT Intron 6 49 281587 281606 1355 532594 CATGACATTGGTTTGGGCAA Intron 6 43 282229 282248 1356 532595 AATGTTGTTGGGAAAATTGG Intron 6 42 282383 282402 1357 532596 AGCTGCAGGATACAAAGTCA Intron 6 49 282986 283005 1358 532597 ATATCCTTTCATGATAAAAA Intron 6 31 283354 283373 1359 532598 ATGGGCTAATATCTCTGATA Intron 6 50 283590 283609 1360 532599 ACATTACTAATAATTAGAGA Intron 6 0 285236 285255 1361 532600 ATAAAAACATATGAAAGTAT Intron 6 12 287093 287112 1362 532601 TTCTGAATTAAATCTATTAG Intron 6 16 287408 287427 1363 532602 TTACATTTTTGCAAATTTAT Intron 6 31 287472 287491 1364 532603 TGAACAGTTGATTAACAAAG Intron 6 15 287887 287906 1365 532604 AAGTTATTGGTTTACTAGAT Intron 6 0 288598 288617 1366 532605 TTGGAAAAGGTCCTAGAAAA Intron 6 24 289808 289827 1367 532606 CATGACAGAAACTTCTTAGA Intron 7 25 292035 292054 1368 532607 CCATACTTGCTGACAAATAT Intron 8 39 294389 294408 1369

Example 115 Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from the studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 141 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523271 41 61 73 86 92 0.8 523274 20 36 64 80 92 1.8 523324 35 45 68 91 90 1.2

TABLE l42 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523604 21 42 68 58 86 2.0 523577 6 22 56 66 91 2.7 523614 14 44 61 84 87 1.9 523564 4 26 48 67 86 2.8 523633 30 43 71 82 84 1.4 523571 2 9 38 55 82 3.9

TABLE 143 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523570 25 50 64 77 88 1.5 523592 27 42 59 79 88 1.7 523595 21 50 62 76 90 1.6 523596 36 47 62 75 77 1.4 523607 49 62 71 82 84 0.5 523615 20 49 63 83 91 1.6 523630 4 28 54 79 78 2.6 523661 4 34 48 73 79 2.7 523665 4 28 54 73 79 2.7 523687 30 56 61 78 81 1.4 523711 42 66 78 94 95 0.7 523712 6 37 60 72 89 2.3 523713 4 32 55 72 85 2.5 523714 59 75 88 95 97 0.2

TABLE 144 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523655 26 33 60 67 78 2.1 523656 19 33 45 69 87 2.4 523658 0 42 62 67 79 3.1 523715 78 90 92 93 95 <0.6 523718 30 46 67 84 92 1.4 523723 56 69 83 92 94 0.3 523725 45 64 79 89 95 0.6 523726 32 48 77 88 89 1.2 523736 0 64 75 90 96 1.5 523747 48 64 80 91 92 0.6 523758 25 39 61 74 84 1.9 523766 7 37 66 81 93 2.0 523776 26 54 72 78 83 1.3 523789 62 68 81 85 90 0.2

TABLE 145 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523719 24 46 65 84 93 1.5 523720 18 49 72 85 93 1.5 523724 43 61 77 91 91 0.7 523735 8 42 63 81 93 2.0 523740 37 58 72 83 88 1.0 523752 9 29 52 72 86 2.5 523763 8 32 57 70 80 2.6 523764 43 52 67 77 79 0.9 523765 24 48 62 88 4 1.5 523767 49 62 67 72 82 0.6 523772 29 39 54 62 61 2.7 523774 28 59 63 88 91 1.2 523778 25 32 63 78 84 1.9 523783 0 22 53 72 88 2.8

TABLE 146 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 532151 57 69 76 85 88 <0.6 532153 23 43 54 80 86 1.8 532158 46 58 81 87 87 0.6 532160 17 26 55 76 92 2.2 532162 14 46 71 83 93 1.7 532164 37 76 82 90 93 0.6 532171 41 81 67 81 83 <0.6 532181 56 81 84 89 93 0.2 532186 26 65 75 83 91 1.1 532188 51 68 80 89 93 <0.6 532189 24 31 52 75 86 2.1 532197 0 40 66 85 93 2.1 532199 24 37 50 73 87 2.1 532222 12 41 67 84 94 1.8

TABLE 147 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 532175 41 54 76 84 89 0.9 532223 53 69 75 88 94 <0.6 532235 43 58 67 77 82 0.8 532241 39 53 62 73 87 1.2 532248 49 65 72 85 93 0.6 532254 52 62 85 87 92 <0.6 532300 20 29 49 66 78 2.7 532304 26 39 66 78 90 1.7 532316 41 66 76 86 94 0.7 532395 32 56 84 93 97 1.0 532401 47 80 92 96 98 <0.6 532411 73 90 94 97 98 <0.6 532420 38 49 82 85 97 1.0 532436 37 58 75 90 96 0.9

TABLE 148 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 532410 66 83 92 94 97 <0.6 532468 45 68 78 93 94 0.6 532469 0 17 56 76 92 2.8 532470 10 34 62 84 94 2.0 532475 13 36 52 64 87 2.5 532476 34 64 73 79 93 0.9 532480 28 54 67 78 87 1.4 532482 21 39 69 83 92 1.7 532490 42 60 68 84 93 0.9 532500 37 50 63 81 87 1.2 532506 13 41 66 75 89 1.9 532507 47 59 71 86 89 0.7 532508 0 31 73 83 89 2.2 532526 31 56 78 79 88 1.1

TABLE 149 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 532495 59 74 81 87 95 <0.6 532501 49 53 71 83 84 0.7 532534 53 75 85 91 97 <0.6 532535 0 34 61 84 92 2.6 532537 49 67 80 90 94 <0.6 532540 59 70 87 93 95 <0.6 532547 57 71 81 91 92 <0.6 532555 48 36 61 72 85 1.3 532556 33 57 67 86 90 1.1

TABLE 150 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523421 32 57 81 82 88 1.0 533006 46 43 69 83 91 1.0 533121 53 75 75 88 93 <0.6 533122 65 77 82 90 93 <0.6 533123 39 71 84 91 95 0.6 533125 49 61 81 85 91 0.6 533131 3 57 59 82 90 1.9 533136 32 65 62 81 88 1.1 533139 13 51 72 90 94 1.5 533140 36 66 39 87 92 1.2 533153 50 65 83 89 90 <0.6 533156 43 64 74 85 90 0.7 533160 57 80 87 91 95 <0.6 533161 54 62 81 89 92 <0.6

TABLE 151 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 533234 50 70 86 93 95 <0.6 533237 5 45 63 84 93 1.9 533233 43 55 76 90 95 0.8 533179 31 63 75 87 87 1.0 533178 53 67 76 89 94 <0.6 533187 5 15 53 79 86 2.7 533188 49 68 83 89 94 <0.6 533271 45 66 85 92 94 0.6 533134 22 45 64 81 89 1.6 533258 52 72 88 93 95 <0.6 533235 50 54 75 82 90 0.7 533262 23 54 78 91 96 1.2 533189 48 66 78 82 88 <0.6 533193 38 53 72 77 91 1.0

TABLE 152 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 533259 63 78 84 90 92 <0.6 533291 25 57 75 86 96 1.2 533256 67 76 90 95 95 <0.6 533269 42 75 82 94 97 0.6 533265 67 78 91 95 97 <0.6 533318 16 45 77 87 95 1.5 533257 55 84 91 96 96 <0.6 533280 34 62 80 91 91 0.9 533301 52 77 84 93 96 <0.6 533316 41 50 79 93 94 0.9 533270 62 71 88 94 97 <0.6 533330 46 76 93 97 98 <0.6 533317 55 60 82 87 96 <0.6 533315 39 56 82 87 93 0.9

TABLE 153 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 533364 71 77 92 90 94 <0.6 533925 26 55 61 85 91 1.4 533326 54 77 80 93 95 <0.6 533916 18 62 69 83 93 1.4 533328 52 68 89 94 98 <0.6 533932 42 49 80 86 92 0.9 533352 42 82 88 93 94 <0.6 533917 20 37 57 78 84 2.0 533331 54 83 89 93 96 <0.6 533936 21 46 73 84 88 1.5 533329 56 73 84 92 98 <0.6 533937 26 32 79 86 94 1.5 533908 58 66 81 88 94 <0.6 533898 61 64 84 90 92 <0.6

TABLE 154 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539371 32 41 82 92 98 1.2 539382 18 58 74 91 97 1.3 539392 34 59 79 94 96 0.9 539398 31 53 89 94 98 1.0 539399 31 72 87 95 97 0.8 539400 36 60 79 93 97 0.9 539405 33 58 74 91 94 1.0 539412 23 61 80 93 95 1.1 539413 53 75 86 92 96 <0.6 539415 47 62 84 91 96 0.6 539416 61 85 94 97 96 <0.6 539430 24 48 68 80 93 1.5 539431 14 40 71 89 95 1.7 539433 46 67 74 92 95 0.6

Example 116 Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from the studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 155 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 523814 0 24 48 52 68 82 2.2 523805 13 29 55 0 79 85 1.5 523822 0 19 26 41 65 85 2.8 523820 0 19 29 58 74 86 2.3 523815 3 6 19 37 45 71 4.8 523828 12 19 32 51 64 74 2.7 523801 3 9 31 43 59 76 3.3 523824 12 28 44 63 77 85 1.7 523794 13 21 30 51 66 78 2.5 523810 15 34 55 72 78 86 1.3 523819 0 24 40 60 66 75 2.4

TABLE 156 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 539302 31 56 80 92 97 98 0.5 539314 16 28 49 69 85 95 1.3 539319 8 30 45 71 90 94 1.4 539320 11 42 64 83 92 95 1.0 539321 25 48 64 82 95 97 0.8 539322 19 34 58 72 90 96 1.1 539331 7 14 46 69 88 96 1.6 539355 28 35 67 89 96 98 0.8 539358 12 39 56 80 93 98 1.1 539359 15 23 58 77 93 98 1.2

TABLE 157 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 539318 23 21 56 73 88 94 1.2 539325 14 26 38 74 92 98 1.4 539339 18 23 58 83 92 98 1.1 539341 17 29 62 84 94 95 1.0 539342 20 31 43 71 90 95 1.2 539352 15 23 41 61 89 95 1.5 539356 24 46 62 83 90 97 0.8 539361 37 42 73 88 96 98 0.6 539379 53 66 83 96 96 98 0.2 539380 52 77 91 97 97 99 0.1 539383 34 61 71 89 98 98 0.5

TABLE 158 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 539360 45 60 81 94 97 98 0.3 539362 21 36 72 90 98 99 0.8 539375 23 36 66 85 95 99 0.9 539376 26 35 58 82 95 99 0.9 539377 29 31 43 64 85 89 1.3 539378 37 59 81 93 97 98 0.4 539389 34 61 61 87 95 97 0.5 539401 34 52 63 84 92 95 0.6 539403 52 73 83 94 97 98 0.1 539404 22 55 74 88 94 96 0.6 539432 32 50 75 86 94 96 0.6

Example 117 Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 159 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523271 26 41 80 89 94 1.4 523274 13 35 63 85 95 1.9 523324 26 40 64 88 95 1.6 523577 27 50 72 87 95 1.3 523604 49 66 74 81 87 0.5 523614 43 54 82 92 89 0.8

TABLE 160 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523564 16 48 69 75 91 1.7 523570 24 52 65 71 88 1.6 523592 6 31 52 65 81 2.8 523595 13 49 60 79 92 1.8 523596 20 49 62 71 75 1.9 523607 38 63 66 74 76 0.8 523615 17 48 60 80 92 1.8 523630 19 42 42 67 80 2.5 523633 41 69 78 79 80 0.6 523665 16 45 56 71 80 2.1 523687 37 59 73 75 78 0.9 523711 33 63 78 91 93 0.9 523712 13 36 61 78 87 2.1 523714 63 85 91 96 96 <0.6

TABLE 161 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523655 28 42 57 74 76 1.9 523656 33 43 53 74 88 1.7 523661 29 29 66 79 82 1.9 523713 35 45 64 83 87 1.3 523715 83 86 92 93 94 <0.6 523718 27 52 69 84 95 1.3 523723 65 74 86 85 94 <0.6 523725 37 63 78 78 92 0.8 523726 43 57 72 86 89 0.8 523736 39 65 80 88 95 0.8 523747 51 71 83 86 93 <0.6 523766 30 50 70 82 89 1.3 523776 45 59 67 79 84 0.7 523789 63 75 76 83 83 <0.6

TABLE 162 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523719 18 40 56 73 83 2.1 523720 36 46 59 64 89 1.5 523724 44 60 75 81 87 0.7 523735 11 40 60 78 84 2.1 523740 17 47 61 80 81 1.8 523752 25 31 38 70 84 2.5 523758 23 48 58 72 80 1.8 523763 2 24 48 64 75 3.3 523764 22 49 45 73 75 2.1 523765 42 40 57 79 87 1.4 523767 43 53 56 69 79 1.2 523774 36 52 71 81 89 1.1 523778 15 45 59 75 79 2.0 523783 5 30 48 66 83 2.9

TABLE 163 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 532151 40 45 64 71 82 1.3 532158 28 47 63 70 87 1.6 532164 36 47 64 75 89 1.3 532171 35 47 50 69 89 1.6 532175 27 38 43 75 87 2.1 532181 21 56 63 69 80 1.7 532186 28 49 62 73 91 1.5 532188 40 52 73 75 90 1.0 532223 22 34 53 71 90 2.2 532235 35 31 48 68 73 2.3 532241 6 24 29 51 72 4.5 532248 19 37 47 73 84 2.3 532254 56 56 72 85 90 0.5 532316 32 55 50 78 90 1.5

TABLE 164 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 532304 44 57 68 78 73 0.7 532395 47 62 82 91 96 0.6 532401 70 83 91 94 96 <0.6 532410 56 71 85 90 96 <0.6 532411 88 93 96 97 98 <0.6 532420 61 67 82 85 96 <0.6 532436 48 49 77 90 97 0.8 532468 42 67 82 89 94 0.6 532476 32 58 75 84 90 1.1 532482 5 26 56 71 87 2.6 532490 18 47 55 69 86 2.0 532501 4 22 43 59 77 3.5 532507 39 63 66 83 89 0.9 532526 30 48 67 82 88 1.4

TABLE 165 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 533121 59 67 78 83 87 0.2 533122 48 73 78 84 90 0.4 533125 47 61 74 89 89 0.6 533136 5 25 58 79 90 2.4 533156 37 48 69 77 87 1.2 533161 28 67 77 89 90 1.0 533178 30 60 72 90 92 1.1 533179 37 66 76 76 87 0.8 533188 32 64 74 80 90 1.0 533189 49 66 77 81 81 0.4 533193 26 48 69 75 85 1.5 533233 39 60 59 84 93 1.0 533234 45 69 84 91 94 0.5 533235 28 49 69 82 90 1.4

TABLE 166 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 533256 47 72 86 90 94 <0.6 533257 63 77 88 91 96 <0.6 533258 66 81 88 95 95 <0.6 533259 48 70 84 90 93 <0.6 533262 44 66 79 90 96 0.7 533265 59 74 85 93 96 <0.6 533269 25 55 74 86 87 1.2 533270 34 59 73 86 95 1.0 533271 63 82 88 92 92 <0.6 533291 14 46 64 84 89 1.8 533301 49 61 75 83 91 0.6 533315 22 39 73 76 91 1.7 533317 26 53 68 85 94 1.3 533318 29 40 46 77 91 1.9

TABLE 167 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 533280 58 64 77 82 87 0.3 533316 35 55 68 87 91 1.1 533326 34 68 76 89 96 0.8 533328 54 55 79 83 92 0.5 533329 46 62 72 83 95 0.7 533330 56 75 83 91 94 0.3 533331 54 61 80 86 89 0.4 533352 54 62 79 83 89 0.4 533364 52 73 83 91 94 0.4 533898 17 47 63 78 87 1.8 533908 35 58 74 82 87 1 533916 22 46 72 78 88 1.6 533932 51 62 70 79 80 0.5 533937 20 40 61 79 85 1.9

Example 118 Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 168 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 523577 0 16 33 59 72 94 2.2 523633 15 33 66 73 82 86 1.1 523764 11 33 50 68 78 83 1.5 523794 12 30 33 56 76 82 1.9 523805 21 48 66 78 85 92 0.8 523810 18 36 61 80 89 90 1.0 523814 13 35 52 67 81 88 1.3 523819 11 30 57 72 81 89 1.3 523820 0 15 43 61 84 92 1.8 523824 21 27 59 72 84 90 1.2

TABLE 169 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 539302 34 41 56 83 83 96 0.8 539321 30 32 76 73 80 94 0.8 539322 22 36 57 72 78 94 1.1 539355 23 42 48 72 71 88 1.2 539359 21 38 48 73 78 92 1.2 539320 14 32 53 72 82 91 1.3 539341 3 19 35 56 78 89 2.0 539342 6 18 33 51 70 83 2.3 539356 0 0 21 45 73 94 2.7 539358 0 15 23 50 52 91 2.9

TABLE 170 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 539339 22 37 52 77 90 92 1.0 539360 28 49 72 82 95 97 0.7 539361 36 56 75 86 95 98 0.5 539362 24 26 63 77 91 97 1.0 539375 21 29 39 63 77 91 1.5 539378  8 42 64 85 92 97 1.0 539379 43 59 80 89 96 98 0.3 539380 61 73 90 95 98 98 0.1 539383 30 49 75 87 97 98 0.6 539403 48 55 75 85 94 96 0.3 539432 36 42 69 79 88 95 0.7

TABLE 171 0.3125 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 539376 34 46 62 82 94 98 0.7 539389 53 58 78 86 94 97 0.2 539392 1 19 26 68 81 94 1.9 539399 27 52 65 78 92 98 0.7 539400 7 26 43 59 88 95 1.6 539401 32 39 77 90 92 95 0.6 539404 22 59 77 87 93 95 0.6 539413 16 33 53 82 86 96 1.1 539415 4 44 56 74 81 94 1.2 539416 37 61 70 85 92 95 0.4 539433 31 52 70 85 87 94 0.6

Example 119 Antisense Inhibition of Human Growth Hormone Receptor in Hep3B Cells by Deoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting a growth hormone receptor (GHR) nucleic acid and were tested for their effects on GHR mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 5,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and (S)-cEt gapmers. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human GHR mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000163.4) or the human GHR genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene.

TABLE 172 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ ID ID NO: 1 NO: 2 SEQ ISIS Start % Start ID NO Site Target Region Sequence Chemistry inhibition Site NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 84 156891 1370 541263 164 Intron 1 CCGAGCTTCGCCTCTG eekddddddddddkke 89   3040 1371 541264 167 Intron 1 CCTCCGAGCTTCGCCT eekddddddddddkke 90   3043 1372 541265 170 Junction GGACCTCCGAGCTTCG eekddddddddddkke 89 n/a 1373 spanning two exons 541266 176 Junction CCTGTAGGACCTCCGA eekddddddddddkke 83 n/a 1374 spanning two exons 541268 214 Exon 2 CCAGTGCCAAGGTCAA eekddddddddddkke 87 144998 1375 541269 226 Exon 2 CACTTGATCCTGCCAG eekddddddddddkke 67 145010 1376 541270 244 Exon 2 CACTTCCAGAAAAAGC eekddddddddddkke 34 145028 1377 541278 365 Exon 4/Intron 3 GTCTCTCGCTCAGGTG eekddddddddddkke 77 268028 1378 541279 368 Exon 4/Intron 3 AAAGTCTCTCGCTCAG eekddddddddddkke 76 268031 1379 541280 373 Exon 4/Intron 3 ATGAAAAAGTCTCTCG eekddddddddddkke 66 268036 1380 541283 445 exon 2-exon 3 TCCTTCTGGTATAGAA eekddddddddddkke 37 n/a 1381 junction 541288 554 Exon 5 CAATAAGGTATCCAGA eekddddddddddkke 49 274114 1382 541289 561 Exon 5 CTTGATACAATAAGGT eekddddddddddkke 66 274121 1383 541290 569 Exon 5 CTAGTTAGCTTGATAC eekddddddddddkke 61 274129 1384 541293 628 exon 3-exon 4 GATCTGGTTGCACTAT eekddddddddddkke 57 n/a 1385 junction 541294 639 Exon 6 GGCAATGGGTGGATCT eekddddddddddkke 38 278933 1386 541295 648 Exon 6 CCAGTTGAGGGCAATG eekddddddddddkke 67 278942 1387 541296 654 Exon 6 TAAAGTCCAGTTGAGG eekddddddddddkke 43 278948 1388 541301 924 Exon 7 TACATAGAGCACCTCA eekddddddddddkke 86 290422 1389 541302 927 Exon 7 TGTTACATAGAGCACC eekddddddddddkke 78 290425 1390 541303 930 Exon 7 AAGTGTTACATAGAGC eekddddddddddkke 59 290428 1391 541304 958 Exon 7 CTTCACATGTAAATTG eekddddddddddkke 26 290456 1392 541305 981 Exon 8 GAGCCATGGAAAGTAG eekddddddddddkke 66 292535 1393 541310 1127 Exon 7-exon 8 CCTTCCTTGAGGAGAT eekddddddddddkke 26 n/a 1394 junction 541320 1317 Exon 10 CTTCACCCCTAGGTTA eekddddddddddkke 38 297734 1395 541321 1322 Exon 10 CCATCCTTCACCCCTA eekddddddddddkke 81 297739 1396 541322 1326 Exon 10 GTCGCCATCCTTCACC eekddddddddddkke 79 297743 1397 541323 1331 Exon 10 CCAGAGTCGCCATCCT eekddddddddddkke 64 297748 1398 541325 1420 Exon 10 GTGGCTGAGCAACCTC eekddddddddddkke 79 297837 1399 541326 1434 Exon 10 CCCTTTTAACCTCTGT eekddddddddddkke 67 297851 1400 541331 1492 Exon 10 CATCATGATAAGGTGA eekddddddddddkke 16 297909 1401 541332 1526 Exon 10 TGGATAACACTGGGCT eekddddddddddkke 30 297943 1402 541333 1532 Exon 10 TCTGCTTGGATAACAC eekddddddddddkke 63 297949 1403 541335 1597 Exon 10 GAATATGGGCAGCTTG eekddddddddddkke 33 298014 1404 541336 1601 Exon 10 AGCTGAATATGGGCAG eekddddddddddkke 34 298018 1405 541337 1607 Exon 10 TTGCTTAGCTGAATAT eekddddddddddkke 39 298024 1406 541338 1611 Exon 10 TGGATTGCTTAGCTGA eekddddddddddkke 79 298028 1407 541339 1614 Exon 10 ACTTGGATTGCTTAGC eekddddddddddkke 73 298031 1408

Example 120 Antisense Inhibition of Human Growth Hormone Receptor in Hep3B Cells by Deoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting a growth hormone receptor (GHR) nucleic acid and were tested for their effects on GHR mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and (S)-cEt gapmers. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human GHR mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000163.4) or the human GHR genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene. The oligonucleotides of Table 175 do not target SEQ ID NOs: 1 or 2, but instead target variant gene sequences SEQ ID NO: 4 (GENBANK Accession No. DR_(006395.1)) or SEQ ID NO: 7 (the complement of GENBANK Accession No. AA398260.1).

TABLE 173 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ ID ID NO: 1 NO: 2 ISIS Start Target % Start SEQ ID NO Site Region Sequence Chemistry inhibition Site NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 86 156891 1370 541340 1619 Exon 10 AGTGAACTTGGATTGC eekddddddddddkke 73 298036 1409 541341 1641 Exon 10 GGCATAAAAGTCGATG eekddddddddddkke 41 298058 1410 541342 1644 Exon 10 CTGGGCATAAAAGTCG eekddddddddddkke 33 298061 1411 541343 1683 Exon 10 GGAAAGGACCACACTA eekddddddddddkke 34 298100 1412 541344 1746 Exon 10 GAGTGAGACCATTTCC eekddddddddddkke 65 298163 1413 541345 1827 Exon 10 GATGTGAGGAGCCACA eekddddddddddkke 54 298244 1414 541346 1830 Exon 10 CTTGATGTGAGGAGCC eekddddddddddkke 70 298247 1415 541347 1835 Exon 10 TCAACCTTGATGTGAG eekddddddddddkke 38 298252 1416 541348 1839 Exon 10 TGATTCAACCTTGATG eekddddddddddkke 39 298256 1417 541349 1842 Exon 10 GTGTGATTCAACCTTG eekddddddddddkke 74 298259 1418 541350 1845 Exon 10 TATGTGTGATTCAACC eekddddddddddkke 58 298262 1419 541351 1949 Exon 10 GGCATCTCAGAACCTG eekddddddddddkke 41 298366 1420 541352 1965 Exon 10 GGTATAGTCTGGGACA eekddddddddddkke 18 298382 1421 541353 1969 Exon 10 TGGAGGTATAGTCTGG eekddddddddddkke 17 298386 1422 541354 1972 Exon 10 GAATGGAGGTATAGTC eekddddddddddkke 0 298389 1423 541355 1975 Exon 10 TATGAATGGAGGTATA eekddddddddddkke 0 298392 1424 541356 1978 Exon 10 CTATATGAATGGAGGT eekddddddddddkke 30 298395 1425 541357 1981 Exon 10 GTACTATATGAATGGA eekddddddddddkke 43 298398 1426 541358 1987 Exon 10 GGGACTGTACTATATG eekddddddddddkke 12 298404 1427 541369 2306 Exon 10 TTACATTGCACAATAG eekddddddddddkke 21 298723 1428 541373 2667 Exon 10 TAGCCATGCTTGAAGT eekddddddddddkke 34 299084 1429 541374 2686 Exon 10 TGTGTAGTGTAATATA eekddddddddddkke 10 299103 1430 541375 2690 Exon 10 ACAGTGTGTAGTGTAA eekddddddddddkke 82 299107 1431 541376 2697 Exon 10 GCAGTACACAGTGTGT eekddddddddddkke 46 299114 1432 541377 2700 Exon 10 ACTGCAGTACACAGTG eekddddddddddkke 32 299117 1433 541378 2740 Exon 10 TTAGACTGTAGTTGCT eekddddddddddkke 25 299157 1434 541379 2746 Exon 10 CCAGCTTTAGACTGTA eekddddddddddkke 69 299163 1435 541380 2750 Exon 10 TAAACCAGCTTTAGAC eekddddddddddkke 20 299167 1436 541381 2755 Exon 10 AACATTAAACCAGCTT eekddddddddddkke 64 299172 1437 541382 2849 Exon 10 ACTACAATCATTTTAG eekddddddddddkke 0 299266 1438 541383 2853 Exon 10 GATTACTACAATCATT eekddddddddddkke 0 299270 1439 541384 2859 Exon 10 AATGCAGATTACTACA eekddddddddddkke 46 299276 1440 541385 2865 Exon 10 TCCAATAATGCAGATT eekddddddddddkke 52 299282 1441 541386 2941 Exon 10 GTTGATCTGTGCAAAC eekddddddddddkke 74 299358 1442 541389 3037 Exon 10 TCTACTTCTCTTAGCA eekddddddddddkke 50 299454 1443 541393 3215 Exon 10 GCTTCTTGTACCTTAT eekddddddddddkke 84 299632 1444 541394 3237 Exon 10 GATTTGCTTCAACTTA eekddddddddddkke 47 299654 1445 541395 3305 Exon 10 GGTTATAGGCTGTGAA eekddddddddddkke 0 299722 1446 541396 3308 Exon 10 TCTGGTTATAGGCTGT eekddddddddddkke 88 299725 1447 541397 3311 Exon 10 GTGTCTGGTTATAGGC eekddddddddddkke 56 299728 1448 541398 3316 Exon 10 AGTATGTGTCTGGTTA eekddddddddddkke 76 299733 1449 541399 3371 Exon 10 GGGACTGAAAACCTTG eekddddddddddkke 50 299788 1450 541400 3975 Exon 10 AGTATTCTTCACTGAG eekddddddddddkke 36 300392 1451 541401 4044 Exon 10 GCGATAAATGGGAAAT eekddddddddddkke 36 300461 1452 541402 4048 Exon 10 GTCTGCGATAAATGGG eekddddddddddkke 52 300465 1453 541403 4058 Exon 10 CCTAAAAAAGGTCTGC eekddddddddddkke 51 300475 1454 541404 4072 Exon 10 CATTAAGCTTGCTTCC eekddddddddddkke 53 300489 1455

TABLE 174 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ ID ID NO: 1 NO: 2 SEQ ISIS Start % Start ID NO Site Target Region Sequence Chemistry inhibition Site NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 85 156891 1370 541421 4418 Exon 10 CACAACTAGTCATACT eekddddddddddkke 42 300835 1456 541422 4428 Exon 10 AACTGCCAGACACAAC eekddddddddddkke 68 300845 1457 541423 4431 Exon 10 ATAAACTGCCAGACAC eekddddddddddkke 86 300848 1458 541424 4503 Exon 10 TATCAGGAATCCAAGA eekddddddddddkke 11 300920 1459 541425 4521 Exon 10 TTGATAACAGAAGCAC eekddddddddddkke 16 300938 1460 541426 4528 Exon 10 TTGGTGTTTGATAACA eekddddddddddkke 31 300945 1461 541427 4531 Exon 10 ATGTTGGTGTTTGATA eekddddddddddkke 32 300948 1462 541429 30 Exon 1 CCGCCACTGTAGCAGC eekddddddddddkke 77 2906 1463 541430 35 Exon 1 CGCCACCGCCACTGTA eekddddddddddkke 88 2911 1464 541431 63 Exon 1 GCCGCCCGGGCTCAGC eekddddddddddkke 86 2939 1465 541432 67 Exon 1 CGCCGCCGCCCGGGCT eekddddddddddkke 61 2943 1466 541433 144 Exon 1 GAGAGCGCGGGTTCGC eekddddddddddkke 57 3020 1467 541434 n/a Exon 1/Intron 1 CTACTGACCCCAGTTC eekddddddddddkke 80 3655 1468 541435 n/a Exon 1/Intron 1 TCACTCTACTGACCCC eekddddddddddkke 90 3660 1469 541436 n/a Exon 1/Intron 1 TCATGCGGACTGGTGG eekddddddddddkke 56 3679 1470 541437 n/a Exon 3/Intron 3 ATGTGAGCATGGACCC eekddddddddddkke 82 225438 1471 541438 n/a Exon 3/Intron 3 TCTTGATATGTGAGCA eekddddddddddkke 93 225445 1472 541439 n/a Exon 3/Intron 3 TTCAAGTTGGTGAGCT eekddddddddddkke 72 226788 1473 541440 n/a Exon 3/Intron 3 TGCTTCCTTCAAGTTG eekddddddddddkke 68 226795 1474 541441 n/a Exon 3/Intron 3 TGTAATTTCATTCATG eekddddddddddkke 62 226809 1475 541442 n/a Exon 3/Intron 3 CCTTTTGCCAAGAGCA eekddddddddddkke 85 226876 1476 541443 n/a Exon 3/Intron 3 GATCCTTTTGCCAAGA eekddddddddddkke 77 226879 1477 541444 n/a Exon 3/Intron 3 GCTAGTAATGTTACAT eekddddddddddkke 68 238331 1478 541445 n/a Exon 3/Intron 3 GCAACTTGCTAGTAAT eekddddddddddkke 65 238338 1479 541446 n/a Exon 3/Intron 3 TGTGCAACTTGCTAGT eekddddddddddkke 44 238341 1480 541447 n/a Exon 3/Intron 3 GGATTTCAGTTTGAAT eekddddddddddkke 0 238363 1481 541448 n/a Exon 3/Intron 3 CTCAGAGCCTTGGTAG eekddddddddddkke 65 238428 1482 541449 n/a Exon 1/Intron 1 CAAACGCGCAAAAGAC eekddddddddddkke 1 3608 1483 541450 n/a Exon 1/Intron 1 GCCCGCACAAACGCGC eekddddddddddkke 11 3615 1484 541451 n/a Exon 1/Intron 1 GGTTAAAGAAGTTGCT eekddddddddddkke 60 93190 1485 541452 n/a Exon 1/Intron 1 CCCAGTGAATTCAGCA eekddddddddddkke 85 93245 1486 541453 n/a Exon 1/Intron 1 GCGCCCAGTGAATTCA eekddddddddddkke 74 93248 1487 541454 n/a Exon 1/Intron 1 AAGATGCGCCCAGTGA eekddddddddddkke 71 93253 1488 541455 n/a Exon 1/Intron 1 TGTAAGATGCGCCCAG eekddddddddddkke 75 93256 1489 541456 n/a Exon 1/Intron 1 AATTACTTGTAAGATG eekddddddddddkke 15 93263 1490 541457 n/a Exon 1/Intron 1 CCCAGAAGGCACTTGT eekddddddddddkke 61 93302 1491 541458 n/a Exon 1/Intron 1 TTGCAGAACAAATCTT eekddddddddddkke 3 93333 1492 541459 n/a Exon 1/Intron 1 CATGGAAGATTTGCAG eekddddddddddkke 17 93343 1493 541460 n/a Exon 1/Intron 1 GGTCATGGAAGATTTG eekddddddddddkke 57 93346 1494 541461 n/a Exon 1/Intron 1 GACCTTGGTCATGGAA eekddddddddddkke 51 93352 1495 541462 n/a Exon 1/Intron 1 TGCCAATCCAAAGAGG eekddddddddddkke 34 93369 1496 541463 n/a Exon 1/Intron 1 GGGTCTGCCAATCCAA eekddddddddddkke 67 93374 1497 541464 n/a Exon 1/Intron 1 TCCCTGGGTCTGCCAA eekddddddddddkke 82 93379 1498 541465 n/a Exon 1/Intron 1 AAGTGTGAATTTATCT eekddddddddddkke 16 93408 1499 541466 n/a Exon 1/Intron 1 GGAGATCTCAACAAGG eekddddddddddkke 38 93428 1500 541468 n/a Exon 1/Intron 1 TCGCCCATCACTCTTC eekddddddddddkke 43 93989 1501 541469 n/a Exon 1/Intron 1 CCTGTCGCCCATCACT eekddddddddddkke 61 93993 1502 541470 n/a Exon 1/Intron 1 TCACCTGTCGCCCATC eekddddddddddkke 70 93996 1503 541471 n/a Exon 1/Intron 1 CCATCACCTGTCGCCC eekddddddddddkke 89 93999 1504 541472 n/a Exon 1/Intron 1 TCACCATCACCTGTCG eekddddddddddkke 72 94002 1505 541473 n/a Exon 1/Intron 1 TAATAGTTGTCACCAT eekddddddddddkke 42 94011 1506 541474 n/a Exon 1/Intron 1 TTCAGATCTTATTAAT eekddddddddddkke 0 94023 1507 541475 n/a Exon 1/Intron 1 TTGCAAATTCAGTCTG eekddddddddddkke 32 94096 1508 541477 n/a Exon 2/Intron 2 CGTTCTCTTGGAAGTA eekddddddddddkke 78 198766 1509 541478 n/a Exon 2/Intron 2 TCTTGAATAAATTTCG eekddddddddddkke 25 198780 1510 541479 n/a Exon 2/Intron 2 AAGCTCACTCTTCAAT eekddddddddddkke 60 198810 1511 541480 n/a Exon 2/Intron 2 TCCAAGCTCACTCTTC eekddddddddddkke 49 198813 1512 541481 n/a Exon 2/Intron 2 GCTCCTGCCACTCTGT eekddddddddddkke 75 198837 1513 541482 n/a Exon 2/Intron 2 ATGGGCAAAGGCATCT eekddddddddddkke 60 198874 1514 541483 n/a 5′ UTR AGTCTTCCCGGCGAGG eekddddddddddkke 32 2571 1515 541484 n/a 5′ and overlappig CCGCCGCTCCCTAGCC eekddddddddddkke 73 2867 1516 with exon 1 541485 n/a Intron 1 GCCCGCAACTCCCTGC eekddddddddddkke 37 3341 1517 541486 n/a Intron 1 CGCCTCCCCAGGCGCA eekddddddddddkke 34 4024 1518 541487 n/a Intron 1 GAGTGTCTTCCCAGGC eekddddddddddkke 86 4446 1519 541488 n/a Intron 1 CTGAAGACTCCTTGAA eekddddddddddkke 39 4721 1520 541489 n/a Intron 1 GGCTAGCCAAGTTGGA eekddddddddddkke 54 5392 1521 541490 n/a Intron 1 TGACTCCAGTCTTACC eekddddddddddkke 76 5802 1522 541491 n/a Intron 1 ATTCATTGTGGTCAGC eekddddddddddkke 91 6128 1523 541492 n/a Intron 1 GAAGTGGGTTTTTCCC eekddddddddddkke 86 6543 1524 541493 n/a Intron 1 GCCTTGGTTCAGGTGA eekddddddddddkke 79 6786 1525

TABLE 175 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting SEQ ID NO: 4 and 7 Target Target SEQ SEQ ISIS Start ID % ID NO Site NO Sequence Chemistry inhibition NO 541428 66 4 CCACTGTAGCAGCCGC eekddddddddddkke 92 1526 541476 263 7 TAGGTATTTCAGAGCC eekddddddddddkke 80 1527

TABLE 176 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic regions of SEQ ID NO: 2 SEQ SEQ ID ID NO: 1 NO: 2 SEQ ISIS Start Start Target % ID NO Site Site Region Sequence Chemistry inhibition NO 541262 156891 541277 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 80 1370 541494 7231 541509 Intron 1 GTCCAGGCAGAGTTGT eekddddddddddkke 30 1528 541495 7570 541510 Intron 1 AGCCAAATGTTGGTCA eekddddddddddkke 19 1529 541496 8395 541511 Intron 1 GAGGGCGAGTTTTTCC eekddddddddddkke 71 1530 541497 9153 541512 Intron 1 GTGGCATTGGCAAGCC eekddddddddddkke 81 1531 541498 9554 541513 Intron 1 ACCCCACTGCACCAAG eekddddddddddkke 67 1532 541499 9931 541514 Intron 1 TCCAAGTACTTGCCAA eekddddddddddkke 83 1533 541500 10549 541515 Intron 1 AGTGCCTGGCCTAAGG eekddddddddddkke 75 1534 541501 11020 541516 Intron 1 GCGCTTCTTCCCTAGG eekddddddddddkke 71 1535 541502 11793 541517 Intron 1 CATCTTGCCCAGGGAT eekddddddddddkke 84 1536 541503 12214 541518 Intron 1 CCATCTTGCTCCAAGT eekddddddddddkke 93 1537 541504 12474 541519 Intron 1 CTTACATCCTGTAGGC eekddddddddddkke 71 1538 541505 12905 541520 Intron 1 CGCCTCCTGGTCCTCA eekddddddddddkke 97 1539 541506 13400 541521 Intron 1 CCCTATGCACTACCTA eekddddddddddkke 49 1540 541507 13717 541522 Intron 1 GAGGGACTGTGGTGCT eekddddddddddkke 65 1541 541508 14149 541523 Intron 1 GCCCAATATGTGCCAG eekddddddddddkke 60 1542 541509 14540 541524 Intron 1 GCTCTCTCATCGCTGG eekddddddddddkke 90 1543 541510 15264 541525 Intron 1 CTCAAGGCTATGTGCC eekddddddddddkke 67 1544 541511 15849 541526 Intron 1 TCCACATCCCTCATGT eekddddddddddkke 68 1545 541512 16530 541527 Intron 1 AGGACTGAAGGCCCAT eekddddddddddkke 49 1546 541513 17377 541528 Intron 1 GTGCGACTTACCAGCT eekddddddddddkke 85 1547 541514 17581 541529 Intron 1 TCGCTAAAGCCACACA eekddddddddddkke 89 1548 541515 17943 541530 Intron 1 GCTCTGGCTGATGGTC eekddddddddddkke 92 1549 541516 18353 541531 Intron 1 TTCCCATGAGGATTTC eekddddddddddkke 70 1550 541517 18636 541532 Intron 1 TTGGGCTTAAGCACTA eekddddddddddkke 71 1551 541518 19256 541533 Intron 1 GCTAGCACCTAGTCCA eekddddddddddkke 71 1552 541519 19814 541534 Intron 1 CCTCTGGCCTACAACA eekddddddddddkke 64 1553 541520 20365 541535 Intron 1 ACCCCTCATCAGCACC eekddddddddddkke 93 1554 541521 20979 541536 Intron 1 GGCCACCCCTGATCCT eekddddddddddkke 66 1555 541522 21566 541537 Intron 1 GAAGCTCCCTTGCCCA eekddddddddddkke 96 1556 541523 22150 541538 Intron 1 AGTGTTGCCCCTCCAA eekddddddddddkke 83 1557 541524 22803 541539 Intron 1 GGGTCTCCAACCTACT eekddddddddddkke 70 1558 541525 29049 541540 Intron 1 GGGATGTAGGTTTACC eekddddddddddkke 74 1559 541526 29554 541541 Intron 1 GCAACCGATATCACAG eekddddddddddkke 60 1560 541527 30245 541542 Intron 1 TGCCCTGGAACAAATT eekddddddddddkke 13 1561 541528 30550 541543 Intron 1 AGTCTAGGAGTAGCTA eekddddddddddkke 50 1562 541529 30915 541544 Intron 1 GCTGTTGTCAAGAGAC eekddddddddddkke 55 1563 541530 31468 541545 Intron 1 CACCTAGACACTCAGT eekddddddddddkke 47 1564 541531 32366 541546 Intron 1 GTCAAGGGATCCCTGC eekddddddddddkke 34 1565 541532 32897 541547 Intron 1 TCCCCCTGGCACTCCA eekddddddddddkke 79 1566 541533 33187 541548 Intron 1 GCCTGGTAACTCCATT eekddddddddddkke 56 1567 541534 33780 541549 Intron 1 GGGCTCACCAACTGTG eekddddddddddkke 39 1568 541535 34407 541550 Intron 1 CCACAGGATCATATCA eekddddddddddkke 37 1569 541536 34846 541551 Intron 1 CTCCAGCAGAAGTGTC eekddddddddddkke 10 1570 541537 35669 541552 Intron 1 AGCCCAACTGTTGCCT eekddddddddddkke 79 1571 541538 36312 541553 Intron 1 TGCCAGGCAGTTGCCA eekddddddddddkke 75 1572 541539 36812 541554 Intron 1 GCCAGTAAGCACCTTG eekddddddddddkke 93 1573 541540 37504 541555 Intron 1 CTAGCTTCCCAGCCCC eekddddddddddkke 46 1574 541541 38841 541556 Intron 1 TCAAGCCCAGCTAGCA eekddddddddddkke 39 1575 541542 39108 541557 Intron 1 CCTCACAGGCCCTAAT eekddddddddddkke 4 1576 541543 39408 541558 Intron 1 ACCTGCTTACATGGTA eekddddddddddkke 21 1577 541544 40250 541559 Intron 1 CCTTTGCTAGGACCCA eekddddddddddkke 52 1578 541545 40706 541560 Intron 1 GGGACTGCCACCAAGG eekddddddddddkke 27 1579 541546 40922 541561 Intron 1 GCTAGATGTTCAGGCC eekddddddddddkke 34 1580 541547 41424 541562 Intron 1 CCTATGGCCATGCTGA eekddddddddddkke 32 1581 541548 41999 541563 Intron 1 GTATGCTAGTTCCCAT eekddddddddddkke 83 1582 541549 42481 541564 Intron 1 CCCTCATAATCTTGGG eekddddddddddkke 13 1583 541550 42700 541565 Intron 1 GTCCAACCACTACCAC eekddddddddddkke 74 1584 541551 43291 541566 Intron 1 ACTTGCAGATAGCTGA eekddddddddddkke 73 1585 541552 43500 541567 Intron 1 GCATGACCCCACTGCC eekddddddddddkke 72 1586 541553 43947 541568 Intron 1 GAGGGTCACATTCCCT eekddddddddddkke 23 1587 541554 44448 541569 Intron 1 TCTCTTACTGGTGGGT eekddddddddddkke 90 1588 541555 45162 541570 Intron 1 GCCCCCTTCCTGGATA eekddddddddddkke 28 1589 541556 46010 541571 Intron 1 CCTCATGCGACACCAC eekddddddddddkke 71 1590 541557 46476 541572 Intron 1 AGCCCTCTGCCTGTAA eekddddddddddkke 67 1591 541558 47447 541573 Intron 1 CTCCCAGCTATAGGCG eekddddddddddkke 38 1592 541559 47752 541574 Intron 1 GCTAGCTGCGCAAGGA eekddddddddddkke 5 1593 541560 48001 541575 Intron 1 GCGCAGCCCGCTGCAA eekddddddddddkke 18 1594 541561 48423 541576 Intron 1 TGCATGATCCACCCCA eekddddddddddkke 65 1595 541562 50195 541577 Intron 1 GCTTAGTGCTGGCCCA eekddddddddddkke 72 1596 541563 50470 541578 Intron 1 CCTTCCAGTCCTCATA eekddddddddddkke 81 1597 541564 51104 541579 Intron 1 ATAGTGTCAAGGCCCA eekddddddddddkke 91 1598 541565 51756 541580 Intron 1 AGGCCTTAGTCACCCA eekddddddddddkke 88 1599 541566 52015 541581 Intron 1 TAACCAACCTAAGGGA eekddddddddddkke 11 1600 541567 52230 541582 Intron 1 ATTCTGGTGATGCCCT eekddddddddddkke 66 1601 541568 52588 541583 Intron 1 GTGTTCACTGCCATGA eekddddddddddkke 67 1602 541569 53532 541584 Intron 1 GGTAGAGCACACTGCC eekddddddddddkke 47 1603 541570 54645 541585 Intron 1 CCACTTTAATGCCACC eekddddddddddkke 76 1604

TABLE 177 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic regions of SEQ ID NO: 2 SEQ SEQ ID ID NO: 2 NO: 2 ISIS Start Stop Target % SEQ ID NO Site Site Region Sequence Chemistry inhibition NO 541262 156891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 88 1370 541571 54886 54901 Intron 1 GTCAAATGCTGTTGGG eekddddddddddkke 91 1605 541572 55900 55915 Intron 1 CATCCCCTATCAGGGT eekddddddddddkke 53 1606 541573 62266 62281 Intron 1 CTCGAATCCCTTGAGC eekddddddddddkke 73 1607 541574 62733 62748 Intron 1 GATTCCCTCCCCTAAC eekddddddddddkke 27 1608 541575 63173 63188 Intron 1 ATCCATCCATGTGCTG eekddddddddddkke 92 1609 541576 63751 63766 Intron 1 GAGCATGCCTCAGTGG eekddddddddddkke 81 1610 541577 63964 63979 Intron 1 CAGAAGGACTGCCTCT eekddddddddddkke 50 1611 541578 64213 64228 Intron 1 ACAATGCTCAACAGCC eekddddddddddkke 75 1612 541579 64576 64591 Intron 1 GTTGGATCTGGCATGC eekddddddddddkke 80 1613 541580 65027 65042 Intron 1 CGGCTGAGAGCAAGGG eekddddddddddkke 88 1614 541581 65363 65378 Intron 1 GAGAGGGTTCAGCCTG eekddddddddddkke 62 1615 541582 65600 65615 Intron 1 ACTTAGTTCCTAGCCA eekddddddddddkke 91 1616 541583 66087 66102 Intron 1 GTGAACCAGATGTGCT eekddddddddddkke 86 1617 541584 66566 66581 Intron 1 GGAGTGACAGCTAAGT eekddddddddddkke 98 1618 541585 66978 66993 Intron 1 AAGTGTTCAGAGCCAC eekddddddddddkke 97 1619 541586 67662 67677 Intron 1 AACCCTGCCAAGGTAC eekddddddddddkke 45 1620 541587 67914 67929 Intron 1 GATGGTGAGCACTACC eekddddddddddkke 78 1621 541588 68278 68293 Intron 1 GGCAGGATAGGACAGA eekddddddddddkke 11 1622 541589 68727 68742 Intron 1 GCAAAGTGATGAGCCT eekddddddddddkke 81 1623 541590 69207 69222 Intron 1 CTATCCACACCATTCC eekddddddddddkke 93 1624 541591 69605 69620 Intron 1 GGATCATGGGCCCCTA eekddddddddddkke 70 1625 541592 70130 70145 Intron 1 GTGAATTTGCTGGGCC eekddddddddddkke 94 1626 541593 70569 70584 Intron 1 GTGATGGGCCCAAGGC eekddddddddddkke 67 1627 541594 71056 71071 Intron 1 TCCTCAGTCGGCTTGC eekddddddddddkke 69 1628 541595 71314 71329 Intron 1 CAGCCTTTTGCCAGAT eekddddddddddkke 93 1629 541596 71620 71635 Intron 1 CCTCCCTAGGATTACC eekddddddddddkke 42 1630 541597 72226 72241 Intron 1 ACGCCCCAATCACTCA eekddddddddddkke 79 1631 541598 72655 72670 Intron 1 GCATGACCCATTATGT eekddddddddddkke 94 1632 541599 73061 73076 Intron 1 TCCCTCCAAGAGCTCA eekddddddddddkke 83 1633 541600 73708 73723 Intron 1 GATGCCTGTGGCTGAC eekddddddddddkke 84 1634 541601 74107 74122 Intron 1 GGCTAGCATGTTGCCT eekddddddddddkke 19 1635 541602 74542 74557 Intron 1 TAACCCACTAGGCTGG eekddddddddddkke 84 1636 541603 74947 74962 Intron 1 TGGCCCAAAACTAATC eekddddddddddkke 34 1637 541604 75192 75207 Intron 1 GGAGCAGTCTGGCACC eekddddddddddkke 85 1638 541605 75699 75714 Intron 1 TATTCTGTGGGACAAG eekddddddddddkke 51 1639 541606 75979 75994 Intron 1 GTGTCTAGTTCCAGCC eekddddddddddkke 86 1640 541607 76410 76425 Intron 1 TACTATCATGTAGCGC eekddddddddddkke 87 1641 541608 76701 76716 Intron 1 TGCCCTTGTAGTGAGA eekddddddddddkke 31 1642 541609 76980 76995 Intron 1 TCCCCAACCTACAAGC eekddddddddddkke 41 1643 541610 77292 77307 Intron 1 GCTCTAGGCATATGAA eekddddddddddkke 63 1644 541611 77555 77570 Intron 1 TACCTCCCTTGTAGGG eekddddddddddkke 27 1645 541612 77854 77869 Intron 1 GGTTCCCTTGCAGAGA eekddddddddddkke 62 1646 541613 78311 78326 Intron 1 GTGCCCTCTTCATGCC eekddddddddddkke 68 1647 541614 79006 79021 Intron 1 CCTGTGTGCAACTGGC eekddddddddddkke 85 1648 541615 79490 79505 Intron 1 CTGAGTCATTTGCCTG eekddddddddddkke 93 1649 541616 79829 79844 Intron 1 GGCCTTAGTAGGCCAG eekddddddddddkke 0 1650 541617 80277 80292 Intron 1 GTCCTTGCAGTCAACC eekddddddddddkke 77 1651 541618 80575 80590 Intron 1 GCTGGGCCAAGTCCAT eekddddddddddkke 77 1652 541619 80895 80910 Intron 1 TAGGGCACTTTTTGCC eekddddddddddkke 31 1653 541620 81207 81222 Intron 1 GCTGAGGTCCCTCTCT eekddddddddddkke 34 1654 541621 81761 81776 Intron 1 CTTTGGTCCCATTGCC eekddddddddddkke 83 1655 541622 82233 82248 Intron 1 GGAACATGCCAAGGGC eekddddddddddkke 91 1656 541623 82738 82753 Intron 1 AGGTGGTCTCCCTTCA eekddddddddddkke 74 1657 541624 83056 83071 Intron 1 TCCCAAAGCTCCCCTC eekddddddddddkke 53 1658 541625 83401 83416 Intron 1 CCTGGCCTAGCAAGCT eekddddddddddkke 47 1659 541626 84048 84063 Intron 1 TCTTAGCCCTGGGCTA eekddddddddddkke 12 1660 541627 84388 84403 Intron 1 GACTTGGACTGGGCTC eekddddddddddkke 81 1661 541628 85261 85276 Intron 1 GGCCTAGGATCTAGGA eekddddddddddkke 0 1662 541629 85714 85729 Intron 1 GTCAGGCTAGAGGGAC eekddddddddddkke 41 1663 541630 86220 86235 Intron 1 GGAAGTTCTCCCAGCC eekddddddddddkke 47 1664 541631 86640 86655 Intron 1 CCTGACTGATGTACAC eekddddddddddkke 35 1665 541632 86903 86918 Intron 1 CTCTGGCCTAGCCTAT eekddddddddddkke 54 1666 541633 87247 87262 Intron 1 GGCTGCTGTCAGATGC eekddddddddddkke 79 1667 541634 88293 88308 Intron 1 TCTCAGGTGTAGGCAG eekddddddddddkke 59 1668 541635 88605 88620 Intron 1 GGTCACTGAGACTGGG eekddddddddddkke 88 1669 541636 88952 88967 Intron 1 ACCCACTAGCAGCTAG eekddddddddddkke 61 1670 541637 89160 89175 Intron 1 CGGATGAGGCAGTTAG eekddddddddddkke 42 1671 541638 89855 89870 Intron 1 TGGTAGGCCCTCTGGC eekddddddddddkke 28 1672 541639 90240 90255 Intron 1 GTCACAAGGTGGGTGC eekddddddddddkke 28 1673 541640 90513 90528 Intron 1 GTCTTGCCCTCACGGA eekddddddddddkke 73 1674 541641 91073 91088 Intron 1 GCAGTCTGTGGACTTA eekddddddddddkke 93 1675 541642 91647 91662 Intron 1 TGCTCTCTGGTCACAC eekddddddddddkke 75 1676 541643 92069 92084 Intron 1 TATCCCCCAGAGCCAT eekddddddddddkke 68 1677 541644 92356 92371 Intron 1 AAGGTGAGAGGGCACT eekddddddddddkke 75 1678 541645 92904 92919 Intron 1 GTTTTAACCTCACCCT eekddddddddddkke 0 1679 541646 93846 93861 Intron 1 CCTTCCACTGACCTTC eekddddddddddkke 56 1680 541647 94374 94389 Intron 1 GACACTAGCCTAAGCC eekddddddddddkke 37 1681

TABLE 178 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic regions of SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 NO: 2 ISIS Start Stop Target % SEQ NO Site Site Region Sequence Chemistry inhibition ID NO 541262 156891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 94 1370 541648 94638 94653 Intron 1 GGTTAGCCCTCAGCCT eekddddddddddkke 61 1682 541649 94839 94854 Intron 1 TATGAAGGTTGGACCA eekddddddddddkke 69 1683 541650 95509 95524 Intron 1 CAACCAGCTCACCTGA eekddddddddddkke 37 1684 541651 95829 95844 Intron 1 GGGCTCCAAGGCTCTC eekddddddddddkke 75 1685 541652 96158 96173 Intron 1 AGCTGTTACATGCCAA eekddddddddddkke 93 1686 541653 96488 96503 Intron 1 GGCCCAGAGGTTATAG eekddddddddddkke 30 1687 541654 96991 97006 Intron 1 GTCCTTAGACCCCTCA eekddddddddddkke 70 1688 541655 97539 97554 Intron 1 GCCCTGGCTAGAGACA eekddddddddddkke 39 1689 541656 98132 98147 Intron 1 CATCCAGCAGCTGGAC eekddddddddddkke 35 1690 541657 98833 98848 Intron 1 GACTGAGGTCATCACA eekddddddddddkke 60 1691 541658 99258 99273 Intron 1 GGCCAGGCACATCATG eekddddddddddkke 45 1692 541659 99843 99858 Intron 1 GGAGCTCATTGAGCCA eekddddddddddkke 36 1693 541660 100406 100421 Intron 1 GTGCCCATTGCTGTGT eekddddddddddkke 70 1694 541661 100742 100757 Intron 1 CCAAGTGTGGCTTCAG eekddddddddddkke 54 1695 541662 101305 101320 Intron 1 CCACCCTTTATACGCA eekddddddddddkke 87 1696 541663 101788 101803 Intron 1 CAGTAACCCCAAGGGA eekddddddddddkke 12 1697 541664 102649 102664 Intron 1 CCCCACCTTATATGGG eekddddddddddkke 9 1698 541665 103034 103049 Intron 1 AGGCCCTTTTTACATG eekddddddddddkke 9 1699 541666 103316 103331 Intron 1 TCAATAAGTCCCTAGG eekddddddddddkke 20 1700 541667 104277 104292 Intron 1 GGCATTGAGTGACTGC eekddddddddddkke 51 1701 541668 104679 104694 Intron 1 ATAATGCCTTCTCAGC eekddddddddddkke 62 1702 541669 106349 106364 Intron 1 GTGAGGCATTTAGCCC eekddddddddddkke 35 1703 541670 106632 106647 Intron 1 GCTCTTGTGTTGGGTA eekddddddddddkke 89 1704 541671 107084 107099 Intron 1 TGTGCAGGAGGTCTCA eekddddddddddkke 60 1705 541672 107949 107964 Intron 1 TGGAGAGTCTTGTCTC eekddddddddddkke 17 1706 541673 108773 108788 Intron 1 GTGACCCACCCAAGAG eekddddddddddkke 34 1707 541674 109336 109351 Intron 1 GTTGTAGCTAGTGTTC eekddddddddddkke 74 1708 541675 109849 109864 Intron 1 GCCTTAGTTTGTGCCA eekddddddddddkke 78 1709 541676 110427 110442 Intron 1 GCCCCAGCTGAGAATT eekddddddddddkke 29 1710 541677 110701 110716 Intron 1 ACAACAATCCAGGGTG eekddddddddddkke 61 1711 541678 110959 110974 Intron 1 CTCCCCTGGAAGTCAC eekddddddddddkke 59 1712 541679 111307 111322 Intron 1 GCCCTCATGGCTCAAG eekddddddddddkke 60 1713 541680 112499 112514 Intron 1 TCAGCAGATAGGGAGC eekddddddddddkke 61 1714 541681 113896 113911 Intron 1 GAATGCGGTGATCAGG eekddddddddddkke 29 1715 541682 117477 117492 Intron 1 CTGAGAGAATTGGCCC eekddddddddddkke 5 1716 541683 117740 117755 Intron 1 AGGCACATTGTTACCA eekddddddddddkke 26 1717 541684 118229 118244 Intron 1 GGGAGGCACTAGAGAA eekddddddddddkke 13 1718 541685 119269 119284 Intron 1 TACAGTAACACATCCC eekddddddddddkke 78 1719 541686 119688 119703 Intron 1 GAAGCTCAGCCTGATC eekddddddddddkke 45 1720 541687 120376 120391 Intron 1 CTTGCCTGACAACCTA eekddddddddddkke 53 1721 541688 120738 120753 Intron 1 GCCTACCTGCTTTTGC eekddddddddddkke 10 1722 541689 121242 121257 Intron 1 TTTCCCAACCACTTAG eekddddddddddkke 7 1723 541690 121615 121630 Intron 1 TCTCCTATTTCAGTTA eekddddddddddkke 23 1724 541691 121823 121838 Intron 1 GGGTGATGGATGAACT eekddddddddddkke 40 1725 541692 122345 122360 Intron 1 ACACTGCTGGTAGTGA eekddddddddddkke 0 1726 541693 122588 122603 Intron 1 ACCCAACTAGCCTGTC eekddddddddddkke 35 1727 541694 123152 123167 Intron 1 GAGACCTGCTGCCTGA eekddddddddddkke 80 1728 541695 123671 123686 Intron 1 ACATCTCTTGGGAGGT eekddddddddddkke 78 1729 541696 124040 124055 Intron 1 ACATAGTACCCCTCCA eekddddddddddkke 35 1730 541697 124430 124445 Intron 1 CTCTCAAGTACCTGCC eekddddddddddkke 72 1731 541698 124824 124839 Intron 1 TTTGTACCCAACCCCC eekddddddddddkke 15 1732 541699 125032 125047 Intron 1 AGGCCCACATAAATGC eekddddddddddkke 21 1733 541700 125533 125548 Intron 1 GAGCATCCCCTACACT eekddddddddddkke 12 1734 541701 126357 126372 Intron 1 GCTGGGCCTTTAGCTG eekddddddddddkke 66 1735 541702 126736 126751 Intron 1 TTGGTCAATTGGGCAG eekddddddddddkke 79 1736 541703 127179 127194 Intron 1 GTCTCATGAGGCCTAT eekddddddddddkke 60 1737 541704 127454 127469 Intron 1 GGAGGTGGGATCCCAC eekddddddddddkke 35 1738 541705 128467 128482 Intron 1 GCCCACTACCTAGCAC eekddddddddddkke 30 1739 541706 129096 129111 Intron 1 CCCAGCTGGCTGGTCG eekddddddddddkke 50 1740 541707 129312 129327 Intron 1 GCACCAGGTCTCCTGT eekddddddddddkke 7 1741 541708 129516 129531 Intron 1 GTCTAGAAGCCTAGGG eekddddddddddkke 23 1742 541709 129976 129991 Intron 1 GCCGGGTGTTGGTGCA eekddddddddddkke 50 1743 541710 130308 130323 Intron 1 TTGGTGCCTGTGTTGC eekddddddddddkke 49 1744 541711 130767 130782 Intron 1 TGCTTCTGATCCCTAC eekddddddddddkke 18 1745 541712 131286 131301 Intron 1 GTTCCCAGGAGGCTTA eekddddddddddkke 56 1746 541713 131676 131691 Intron 1 AGGCCCCTAGAGTCTA eekddddddddddkke 41 1747 541714 132292 132307 Intron 1 TGGTGTGCCCAGACTT eekddddddddddkke 60 1748 541715 132730 132745 Intron 1 GATGGCTAACCCACTG eekddddddddddkke 14 1749 541716 133101 133116 Intron 1 CCCCCAAAAGTTGCCC eekddddddddddkke 12 1750 541717 133522 133537 Intron 1 TAGGGTGTTCCAGATC eekddddddddddkke 44 1751 541718 133724 133739 Intron 1 GTACCATGAAGCTCTG eekddddddddddkke 67 1752 541719 134086 134101 Intron 1 CTTGGACTTGGACCAT eekddddddddddkke 42 1753 541720 134441 134456 Intron 1 GTGCATAGGGCCTGTC eekddddddddddkke 42 1754 541721 135015 135030 Intron 1 CCTCACCTGAACACCC eekddddddddddkke 23 1755 541722 135859 135874 Intron 1 ATGCCTCCCCGCAACT eekddddddddddkke 27 1756 541723 136287 136302 Intron 1 TTGTGCTTGGGTGTAC eekddddddddddkke 39 1757 541724 137000 137015 Intron 1 AGGCTTCATGTGAGGT eekddddddddddkke 86 1758

TABLE 179 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting introns 1 and 2 of SEQ ID NO: 2 SEQ SEQ ID NO: ID NO: ISIS 2 Start 2 Stop Target % SEQ NO Site Site Region Sequence Chemistry inhibition ID NO 541262 156891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 95 1370 541725 137372 137387 Intron 1 TGTAAAAGGTCCTCCC eekddddddddddkke 53 1759 541726 137750 137765 Intron 1 GACCTGTGCAGCAGGT eekddddddddddkke 32 1760 541727 138783 138798 Intron 1 TCCTCTTGGAGATCCA eekddddddddddkke 44 1761 541728 139825 139840 Intron 1 AGGTCATAGGACTGCT eekddddddddddkke 73 1762 541729 140343 140358 Intron 1 GAAGGTCAGACTAGGG eekddddddddddkke 53 1763 541730 140686 140701 Intron 1 TCTGTAGACTGCCCAG eekddddddddddkke 87 1764 541731 141116 141131 Intron 1 GTCCCTCTATTCCCCT eekddddddddddkke 57 1765 541732 141591 141606 Intron 1 AATTGCCATGCTCCCA eekddddddddddkke 56 1766 541733 142113 142128 Intron 1 GATGACCTTCCTCCAA eekddddddddddkke 15 1767 541734 142327 142342 Intron 1 GTTTCCAGTAGCACCT eekddddddddddkke 82 1768 541735 143118 143133 Intron 1 GGCCTTGAGCTGATGG eekddddddddddkke 11 1769 541736 143836 143851 Intron 1 TATCCCTAATCAGGCT eekddddddddddkke 40 1770 541737 144094 144109 Intron 1 GGTGTCCACATCCCGG eekddddddddddkke 58 1771 541738 144558 144573 Intron 1 AGCTGGACAGGCCATA eekddddddddddkke 27 1772 541740 145510 145525 Intron 2 GGTAATCACCCAGAGA eekddddddddddkke 90 1773 541741 145937 145952 Intron 2 GCGCTAAGTCTGCTGT eekddddddddddkke 92 1774 541742 146320 146335 Intron 2 CCTCAAATCTTGCCCA eekddddddddddkke 96 1775 541743 147028 147043 Intron 2 ATCCAGACCTGGCAGA eekddddddddddkke 84 1776 541744 147262 147277 Intron 2 ATCCCTGCTCAAGTGC eekddddddddddkke 89 1777 541745 147671 147686 Intron 2 CAGGCACTCCTTGGAA eekddddddddddkke 93 1778 541746 148139 148154 Intron 2 AGCTGAGGTATCCCTC eekddddddddddkke 94 1779 541747 148564 148579 Intron 2 GGGCCCAGCAAGTCTT eekddddddddddkke 33 1780 541748 149069 149084 Intron 2 GTTTTGTCAGTGTGCA eekddddddddddkke 98 1781 541749 149491 149506 Intron 2 GTGACCTGCTGAACTC eekddddddddddkke 95 1782 541750 150236 150251 Intron 2 GGCTGAACTGTGCACC eekddddddddddkke 95 1783 541751 150748 150763 Intron 2 GGGTGGTCCCACTCCT eekddddddddddkke 91 1784 541752 151124 151139 Intron 2 GAGGAATCCTGGGCCC eekddddddddddkke 94 1785 541753 151373 151388 Intron 2 ATGACAAGCTAGGTGC eekddddddddddkke 81 1786 541754 151644 151659 Intron 2 TTGCCAGACAGGGCAC eekddddddddddkke 18 1787 541755 152373 152388 Intron 2 AGACCCCTCCCACTAT eekddddddddddkke 43 1788 541756 152617 152632 Intron 2 GGTGCTGGGTGACCGG eekddddddddddkke 91 1789 541757 153349 153364 Intron 2 GGCCAAACGGTGCCCT eekddddddddddkke 23 1790 541758 153918 153933 Intron 2 TGGGTGAATAGCAACC eekddddddddddkke 85 1791 541759 154171 154186 Intron 2 GCCCCCAAGGAAGTGA eekddddddddddkke 76 1792 541760 154813 154828 Intron 2 CAGGCTTCATGTGTGG eekddddddddddkke 92 1793 541761 155289 155304 Intron 2 CTGTCAGTGCTTTGGT eekddddddddddkke 52 1794 541762 156233 156248 Intron 2 GAGTACCCTGGCAGGT eekddddddddddkke 58 1795 541763 156847 156862 Intron 2 TAGCTAGCACCTGGGT eekddddddddddkke 90 1796 541764 157552 157567 Intron 2 GGCAAACCTTTGAGCC eekddddddddddkke 27 1797 541765 157927 157942 Intron 2 GCTATCATTGGAGCAG eekddddddddddkke 94 1798 541766 158542 158557 Intron 2 CCTCTGAGTACTCCCT eekddddddddddkke 96 1799 541767 159252 159267 Intron 2 AGCTGAAGGCAACCAG eekddddddddddkke 97 1800 541768 159539 159554 Intron 2 GGGCAGTTTTCCATAG eekddddddddddkke 89 1801 541769 159778 159793 Intron 2 GGTCCTACCTCTGACA eekddddddddddkke 82 1802 541770 160352 160367 Intron 2 GGCTGCCTTAGGGTGG eekddddddddddkke 90 1803 541771 160812 160827 Intron 2 CGCACCTCCCCCACTA eekddddddddddkke 15 1804 541772 161461 161476 Intron 2 GCTTATTGGTCCATGG eekddddddddddkke 93 1805 541773 161821 161836 Intron 2 AACCGCAGAGCCCCCA eekddddddddddkke 76 1806 541774 162132 162147 Intron 2 GGGCTTGTTCTGCCAA eekddddddddddkke 33 1807 541775 162639 162654 Intron 2 GGGACCTGCGCTGACT eekddddddddddkke 86 1808 541776 163024 163039 Intron 2 CTTTCACCTGGTGACT eekddddddddddkke 83 1809 541777 163542 163557 Intron 2 AGCTTGAGGGAGTATA eekddddddddddkke 52 1810 541778 164144 164159 Intron 2 GCCTGCTCAATTGAGG eekddddddddddkke 32 1811 541779 164570 164585 Intron 2 ATAGCAGCTGGCTGCC eekddddddddddkke 24 1812 541780 165419 165434 Intron 2 AAAAGCTTGGCACCCC eekddddddddddkke 91 1813 541781 165859 165874 Intron 2 CCTGGCAAGAAGGGCC eekddddddddddkke 65 1814 541782 166435 166450 Intron 2 TTAGCCCATCTATCCC eekddddddddddkke 82 1815 541783 166837 166852 Intron 2 GTGGTCTCCCTGTGCC eekddddddddddkke 90 1816 541784 167107 167122 Intron 2 AGCCCTCTCTGGCAAA eekddddddddddkke 38 1817 541785 168004 168019 Intron 2 TTACTGTGGCCCGAGT eekddddddddddkke 94 1818 541786 169062 169077 Intron 2 GTAGACTCCTAGGGTC eekddddddddddkke 90 1819 541787 169696 169711 Intron 2 CCTCCAGTTAGTGTGC eekddddddddddkke 91 1820 541788 170081 170096 Intron 2 GTGGGTGGCCAACAGG eekddddddddddkke 91 1821 541789 170799 170814 Intron 2 GGGATTCCCTGGTAGC eekddddddddddkke 77 1822 541790 171021 171036 Intron 2 GTGAGACCGGCCTTTG eekddddddddddkke 23 1823 541791 171530 171545 Intron 2 ACTGGCACCCACTTGG eekddddddddddkke 54 1824 541792 172447 172462 Intron 2 ATTGGCCTAATGCCCC eekddddddddddkke 76 1825 541793 172733 172748 Intron 2 AGGCTATACATTCCAG eekddddddddddkke 94 1826 541794 173045 173060 Intron 2 GGTGGCAGCTAGGTGG eekddddddddddkke 80 1827 541795 173677 173692 Intron 2 TCCACAGTTGGCACTG eekddddddddddkke 77 1828 541796 174128 174143 Intron 2 TGGGCCTTAGATTGTA eekddddddddddkke 69 1829 541797 174521 174536 Intron 2 TGTCTTCCTGGTGGCC eekddddddddddkke 97 1830 541798 174870 174885 Intron 2 CCCGCCTCTCCAGCAA eekddddddddddkke 89 1831 541799 175275 175290 Intron 2 GCAGCAGCCAATAAGT eekddddddddddkke 76 1832 541800 175691 175706 Intron 2 TTGTATCCTGGCCCCT eekddddddddddkke 80 1833 541801 176038 176053 Intron 2 GCCTCATGGGCCTTAC eekddddddddddkke 66 1834

TABLE 180 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ ID ID NO: NO: 2 SEQ ISIS 2 Start Stop Target % ID NO Site Site Region Sequence Chemistry inhibition NO 541262 156891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 97 1370 541802 176619 176634 Intron 2 GGATGCCAGTCTTGGC eekddddddddddkke 48 1835 541803 176835 176850 Intron 2 CTGCTCTCAGTACCTC eekddddddddddkke 87 1836 541804 177300 177315 Intron 2 ACCCAAGAAGTCACCT eekddddddddddkke 93 1837 541805 177551 177566 Intron 2 GCCTCAAGCCCTACCC eekddddddddddkke 73 1838 541806 178066 178081 Intron 2 AGCTCCAGCCTATAGA eekddddddddddkke 81 1839 541807 178361 178376 Intron 2 GGTCCACATGGCCCTA eekddddddddddkke 90 1840 541808 178895 178910 Intron 2 CAGGCCCAGGATTGTC eekddddddddddkke 81 1841 541809 179444 179459 Intron 2 GGGCCTGCTTTGCAGC eekddddddddddkke 81 1842 541810 179863 179878 Intron 2 ACTCCTCTCTTTAGGC eekddddddddddkke 87 1843 541811 180524 180539 Intron 2 CTGGGTAACAGTCCTC eekddddddddddkke 98 1844 541812 181528 181543 Intron 2 ACTGTATGGTTTCCAC eekddddddddddkke 83 1845 541813 182103 182118 Intron 2 GCCAAAGATAGCTCTT eekddddddddddkke 94 1846 541814 182978 182993 Intron 2 GGCATTGGAAGTTGGT eekddddddddddkke 87 1847 541815 183193 183208 Intron 2 CCCTTCCTGACCTTAC eekddddddddddkke 55 1848 541816 183658 183673 Intron 2 TTACCCTCTATTCACC eekddddddddddkke 65 1849 541818 184501 184516 Intron 2 GGCACCCCAGGCCGGG eekddddddddddkke 25 1850 541819 185080 185095 Intron 2 CAGCAGCTAGTTCCCC eekddddddddddkke 96 1851 541820 185327 185342 Intron 2 GTGGGCACTAGTGTGT eekddddddddddkke 75 1852 541821 185682 185697 Intron 2 TGCCCTTGTCAGGGCA eekddddddddddkke 20 1853 541822 186025 186040 Intron 2 GCAGATAGGCTCAGCA eekddddddddddkke 98 1854 541823 186570 186585 Intron 2 CCCTAGCCCTTAGCAC eekddddddddddkke 44 1855 541824 186841 186856 Intron 2 ACTGGAATGGCCCTCT eekddddddddddkke 86 1856 541825 187176 187191 Intron 2 TTTGCTCATGCTCACA eekddddddddddkke 96 1857 541826 187629 187644 Intron 2 GCCTTTGTGTGTCACT eekddddddddddkke 99 1858 541827 187857 187872 Intron 2 TATGTGGTAGCATGTC eekddddddddddkke 96 1859 541828 188442 188457 Intron 2 CCCCAGGAAGTTGGCC eekddddddddddkke 68 1860 541829 189086 189101 Intron 2 TAGCTGTCAAGGCCCT eekddddddddddkke 90 1861 541830 189534 189549 Intron 2 CCTAGTCAGCCACTAG eekddddddddddkke 20 1862 541831 189889 189904 Intron 2 AGACTCCCCATCAGCC eekddddddddddkke 74 1863 541832 190172 190187 Intron 2 GTGAAGGGCCTTCATC eekddddddddddkke 68 1864 541833 190961 190976 Intron 2 GGTTGAGAGTCCAATG eekddddddddddkke 95 1865 541834 191404 191419 Intron 2 CAGCTAATTCCCTCAT eekddddddddddkke 79 1866 541835 191614 191629 Intron 2 TTGTGTCTCAACCCAC eekddddddddddkke 95 1867 541836 191999 192014 Intron 2 GGCTATGCTGCATGCT eekddddddddddkke 91 1868 541837 192860 192875 Intron 2 CCCCATACCCAGTGGA eekddddddddddkke 71 1869 541838 193460 193475 Intron 2 GGTGGTTTTCCTCCCT eekddddddddddkke 95 1870 541839 194144 194159 Intron 2 GAGCCTGCCCAACTTT eekddddddddddkke 90 1871 541840 194425 194440 Intron 2 TGATGCCCAAGAGTGA eekddddddddddkke 85 1872 541841 194953 194968 Intron 2 TTCCCTCTGCGAACAT eekddddddddddkke 96 1873 541842 195428 195443 Intron 2 GTTCCATCTCAATCCA eekddddddddddkke 94 1874 541843 196858 196873 Intron 2 ACGGCCACTCCACTGG eekddddddddddkke 44 1875 541844 197326 197341 Intron 2 TGGAAGTGGTTCCAGA eekddddddddddkke 90 1876 541845 197946 197961 Intron 2 TTGCCCCAGACCAACA eekddddddddddkke 47 1877 541846 198366 198381 Intron 2 GAGGTTGTGGAGGTGC eekddddddddddkke 26 1878 541847 198715 198730 Intron 2 GAGTTGCTGTGTGTGA eekddddddddddkke 83 1879 541848 198939 198954 Intron 2 CATGTCAGAGGTGTCC eekddddddddddkke 93 1880 541849 199506 199521 Intron 2 AGGTAAGGATCATGGC eekddddddddddkke 87 1881 541850 199816 199831 Intron 2 GTTCAGTTGCATCACG eekddddddddddkke 90 1882 541851 200249 200264 Intron 2 GCCCAGCTAGCCACCC eekddddddddddkke 68 1883 541852 201258 201273 Intron 2 CCTTAGCAGCCAGGCC eekddddddddddkke 86 1884 541853 202079 202094 Intron 2 GCACTTAGGGTTTTGC eekddddddddddkke 94 1885 541854 202382 202397 Intron 2 GTTGAACTTTCCCTAC eekddddddddddkke 53 1886 541855 202702 202717 Intron 2 TGACTCCTTGAGACAG eekddddddddddkke 83 1887 541856 203098 203113 Intron 2 TGCGCTGGCTTAGCAA eekddddddddddkke 59 1888 541857 203464 203479 Intron 2 GGCCTAACATCAGCAG eekddddddddddkke 88 1889 541858 204212 204227 Intron 2 ACTCCTCCCAGTTAGC eekddddddddddkke 70 1890 541859 205630 205645 Intron 2 ACCAGTGGCCAATGTC eekddddddddddkke 92 1891 541861 206422 206437 Intron 2 GCCTAGACACAGTAGG eekddddddddddkke 70 1892 541862 206749 206764 Intron 2 TATTCTCCCCCTAGGG eekddddddddddkke 42 1893 541863 207517 207532 Intron 2 GACGGCCTTGGGCACA eekddddddddddkke 96 1894 210196 210211 541865 208659 208674 Intron 3 GCAGGCTGTATTAGCA eekddddddddddkke 15 1895 541867 209999 210014 Intron 3 ACCCCCTATCCTGCAC eekddddddddddkke 58 1896 541868 210281 210296 Intron 3 TCCTCCATACCTAGAG eekddddddddddkke 61 1897 211033 211048 541869 210502 210517 Intron 3 GATAGGTGCCCACTGT eekddddddddddkke 80 1898 541870 210920 210935 Intron 3 GTCAGTTCTGGCTAGG eekddddddddddkke 97 1899 541871 211269 211284 Intron 3 GCCTGAACTTACAAGC eekddddddddddkke 68 1900 541872 211836 211851 Intron 3 ACCCTGGGCTGACCTT eekddddddddddkke 92 1901 541873 212606 212621 Intron 3 GGACCTGGACAAGCAA eekddddddddddkke 97 1902 541874 213099 213114 Intron 3 CTCCTTGCGAGAGAGG eekddddddddddkke 7 1903 541875 213425 213440 Intron 3 AGAGTTGACATGGGCA eekddddddddddkke 96 1904 541876 213846 213861 Intron 3 CACTAGGTCCCTGACC eekddddddddddkke 37 1905 541877 214483 214498 Intron 3 CACTCTCTTGGGCTGT eekddddddddddkke 94 1906 541878 214884 214899 Intron 3 AGGGACCTGCATTCCA eekddddddddddkke 72 1907

TABLE 181 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ ID ID NO: ISIS NO: 2 2 Stop Target % SEQ ID NO Start Site Site Region Sequence Chemistry inhibition NO 541262 156891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 91 1370 541879 215493 215508 Intron 3 TTCACCACCCATTGGG eekddddddddddkke 63 1908 541880 216192 216207 Intron 3 ATCTGGTCTGAGGGCC eekddddddddddkke 92 1909 541881 216458 216473 Intron 3 GACATGCAATTGACCC eekddddddddddkke 98 1910 541882 217580 217595 Intron 3 GTGTGCAGCAGACTGT eekddddddddddkke 92 1911 541883 218233 218248 Intron 3 GACAGTCCAGCTGCAA eekddddddddddkke 84 1912 541884 218526 218541 Intron 3 CCTGCGGCAGTGAAGA eekddddddddddkke 85 1913 541885 218734 218749 Intron 3 CTCTGAGGATAACCCT eekddddddddddkke 76 1914 541886 219342 219357 Intron 3 GTTCCCAGCTCCCCAA eekddddddddddkke 68 1915 541887 219618 219633 Intron 3 TAGGGTCAGTGTCCCA eekddddddddddkke 79 1916 541888 220039 220054 Intron 3 GGCGAGCCTCTCAGCC eekddddddddddkke 52 1917 541889 220393 220408 Intron 3 GACTCATCCAGGCAGT eekddddddddddkke 91 1918 541890 220665 220680 Intron 3 TCCCTCCCTTAGGCAC eekddddddddddkke 71 1919 541891 221044 221059 Intron 3 GAGGAGCCAGGCATAT eekddddddddddkke 80 1920 541892 221562 221577 Intron 3 CACCAACGAAGTCCCC eekddddddddddkke 89 1921 541893 221947 221962 Intron 3 GCTGGCAGTCACCAAA eekddddddddddkke 90 1922 541894 222569 222584 Intron 3 GCCCACACCATTGAGC eekddddddddddkke 70 1923 541895 222983 222998 Intron 3 AGTGAGATGCCCTGGT eekddddddddddkke 92 1924 541896 223436 223451 Intron 3 CACTGGCAGTTAGACC eekddddddddddkke 88 1925 541897 224107 224122 Intron 3 ACTCTGGCCACTAGTA eekddddddddddkke 80 1926 541898 224731 224746 Intron 3 GGTAGGGTGGCCACAT eekddddddddddkke 78 1927 541899 225133 225148 Intron 3 GAGCCATGTCTAGGCA eekddddddddddkke 18 1928 541900 225465 225480 Intron 3 CAGACTGAAACCCACC eekddddddddddkke 86 1929 541901 225671 225686 Intron 3 TATGGTCCAGCCACCA eekddddddddddkke 76 1930 541902 226110 226125 Intron 3 TACCTCCTCTGTTGGT eekddddddddddkke 36 1931 541903 227025 227040 Intron 3 ACACCTCAGTCATGAT eekddddddddddkke 92 1932 541904 227236 227251 Intron 3 AACAGGCTTCAAGAGG eekddddddddddkke 91 1933 541905 227485 227500 Intron 3 GTACTACTGGCCATGT eekddddddddddkke 73 1934 541906 227914 227929 Intron 3 CTGCAGGCGGTTGCTA eekddddddddddkke 60 1935 541907 228718 228733 Intron 3 GTCTGTTGCCAAGAGC eekddddddddddkke 95 1936 541908 229174 229189 Intron 3 CCCTGGGTCACTTAAG eekddddddddddkke 44 1937 541909 229423 229438 Intron 3 CCTGTCCTTGCTTGCA eekddddddddddkke 96 1938 541910 230042 230057 Intron 3 GCCCAGCTTATCCTAA eekddddddddddkke 78 1939 541911 230313 230328 Intron 3 AGTAGAGCCTTTGCCT eekddddddddddkke 75 1940 541912 230580 230595 Intron 3 CTGTCTCTTGGCCCAT eekddddddddddkke 80 1941 541913 231330 231345 Intron 3 GGCCCAAATCTTGAGT eekddddddddddkke 67 1942 541914 231817 231832 Intron 3 GCTTGTTACAGCACTA eekddddddddddkke 92 1943 541915 232088 232103 Intron 3 ACTTTGGCCCAGAGAT eekddddddddddkke 51 1944 541916 232884 232899 Intron 3 GCAGTCAGGTCAGCTG eekddddddddddkke 75 1945 541917 233210 233225 Intron 3 GCCTTGTCCTACTACC eekddddddddddkke 65 1946 541918 233657 233672 Intron 3 GGCTCTGCTATTGGCC eekddddddddddkke 59 1947 541919 233998 234013 Intron 3 CTTATAGAGCCTTGCC eekddddddddddkke 59 1948 541920 234296 234311 Intron 3 GGAAGGGCCCAAATAT eekddddddddddkke 15 1949 541921 234903 234918 Intron 3 GATCTACTCCTACTGC eekddddddddddkke 65 1950 541922 235313 235328 Intron 3 GTCAGCCTGTGTCTGA eekddddddddddkke 45 1951 541923 235770 235785 Intron 3 AGCTTCCTCCTTACAC eekddddddddddkke 54 1952 541924 236198 236213 Intron 3 CTGCTAAGCCCCTACC eekddddddddddkke 59 1953 541925 236684 236699 Intron 3 AGAGGTCAGGTGCATA eekddddddddddkke 77 1954 541926 237055 237070 Intron 3 TTCAGCCTGGTTGGGA eekddddddddddkke 71 1955 541927 237585 237600 Intron 3 GATTGATTGAGCTCCT eekddddddddddkke 86 1956 541928 237949 237964 Intron 3 ATGGACTCCCTAGGCT eekddddddddddkke 61 1957 541929 238542 238557 Intron 3 TACTCAAGGGCCCCTC eekddddddddddkke 67 1958 541930 245319 245334 Intron 3 GGCATATGTAGCTTGC eekddddddddddkke 91 1959 541931 245765 245780 Intron 3 GAGCTTAGATCTGTGC eekddddddddddkke 73 1960 541932 246251 246266 Intron 3 ATGCTCACGGCTGTGT eekddddddddddkke 81 1961 541933 246500 246515 Intron 3 ATTGAAAGGCCCATCA eekddddddddddkke 45 1962 541934 246936 246951 Intron 3 CAACCCAGTTTGCCGG eekddddddddddkke 71 1963 541935 247225 247240 Intron 3 CAGCTATTCCCTGTTT eekddddddddddkke 53 1964 541936 247644 247659 Intron 3 GCTGTGTCACACTTCC eekddddddddddkke 98 1965 541937 248223 248238 Intron 3 GTCCAAGGATCACAGC eekddddddddddkke 86 1966 541938 248695 248710 Intron 3 GCTACCACTAGAGCCT eekddddddddddkke 81 1967 541939 249494 249509 Intron 3 GTTTCAGGGCTTATGT eekddddddddddkke 63 1968 541940 250693 250708 Intron 3 TCCCACACCTATTGAA eekddddddddddkke 51 1969 541941 251622 251637 Intron 3 ACTGACTAGAGAGTCC eekddddddddddkke 81 1970 541942 251950 251965 Intron 3 TCCAAGGCTGATGTCC eekddddddddddkke 85 1971 541943 252665 252680 Intron 3 TCCCATGGTGGACATG eekddddddddddkke 39 1972 541944 253140 253155 Intron 3 AGTAGCTGGCAGAAGG eekddddddddddkke 85 1973 541945 253594 253609 Intron 3 CTGGGAGTGACTACTA eekddddddddddkke 77 1974 541946 254036 254051 Intron 3 TGGTATAGCTACTGGG eekddddddddddkke 84 1975 541947 254905 254920 Intron 3 CTGTGGTTTGGCAGGT eekddddddddddkke 90 1976 541948 255407 255422 Intron 3 GTTCTCACCTGAACTA eekddddddddddkke 65 1977 541949 255618 255633 Intron 3 ATAGGCTACTGGCAGG eekddddddddddkke 89 1978 541950 255992 256007 Intron 3 CCCAGCTAGCTGGAGT eekddddddddddkke 50 1979 541951 256428 256443 Intron 3 GGCTGGCTCTCAAAGG eekddddddddddkke 61 1980 541952 256689 256704 Intron 3 TGGTGATACTGTGGCA eekddddddddddkke 94 1981 541953 257317 257332 Intron 3 GCTGATTTTGGTGCCA eekddddddddddkke 92 1982 541954 257826 257841 Intron 3 GCTAATCTTGCCTCGA eekddddddddddkke 52 1983 541955 258407 258422 Intron 3 CACTGGTGGCTTTCAA eekddddddddddkke 31 1984

TABLE 182 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 SEQ SEQ ID ID NO: NO: 2 SEQ ISIS 1 Start Target % Start ID NO Site Region Sequence Chemistry inhibition Site NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 93 156891 1370 541956 n/a Intron 3 GTCCCCTTCTTAAGCA eekddddddddddkke 56 258980 1985 541957 n/a Intron 3 GCCAGGCCAACTGTGG eekddddddddddkke 53 259290 1986 541958 n/a Intron 3 GGCCCGTTATGGTGGA eekddddddddddkke 72 259500 1987 541959 n/a Intron 3 CCTAAAGTCCAACTCC eekddddddddddkke 76 261641 1988 541960 n/a Intron 3 CCCTATCCAGCCTTCA eekddddddddddkke 77 262021 1989 541961 n/a Intron 3 AAGCATGGCCTCTGGC eekddddddddddkke 23 262453 1990 541962 n/a Intron 3 TACCCTGCACCCTCCT eekddddddddddkke 71 262764 1991 541963 n/a Intron 3 TCCTTAGTAGAATGCC eekddddddddddkke 82 263342 1992 541964 n/a Intron 3 TTAGCCCTGGGAGCAC eekddddddddddkke 78 263913 1993 541965 n/a Intron 3 GCTGGGTCAGGTAGCG eekddddddddddkke 71 266503 1994 541966 n/a Intron 3 GGGAGGCTCTCAATCT eekddddddddddkke 75 266861 1995 541967 n/a Intron 3 GTAAGTGCAGAATGCC eekddddddddddkke 87 267116 1996 541968 n/a Intron 3 TGCCGAGGCAGGCACC eekddddddddddkke 33 267380 1997 541969 n/a Intron 3 TCCGTGTCTAGGAGGT eekddddddddddkke 84 267865 1998 541970 n/a Intron 4 GTCTCCCTGCATTGGA eekddddddddddkke 31 268366 1999 541971 n/a Intron 4 CCATATCACTCTCCTC eekddddddddddkke 79 268786 2000 541972 n/a Intron 4 CGAACACCTTGAGCCA eekddddddddddkke 90 269252 2001 541973 n/a Intron 4 GGCCCAGCTTAAGAGG eekddddddddddkke 59 270038 2002 541974 n/a Intron 4 CTGATACTCCTAATCC eekddddddddddkke 70 270501 2003 541975 n/a Intron 4 GCCTGTAGGGCTGTGC eekddddddddddkke 82 270817 2004 541976 n/a Intron 4 TGCCCTTTCTCCCTAC eekddddddddddkke 87 271216 2005 541977 n/a Intron 4 AGTGCATGTCAGTACC eekddddddddddkke 75 271812 2006 541978 n/a Intron 4 TGCTCCTCAGCTGTTG eekddddddddddkke 44 272631 2007 541979 n/a Intron 4 GTTTGGGACCATCCCT eekddddddddddkke 41 272834 2008 541980 n/a Intron 4 AGTGCTCTCTAGGGTC eekddddddddddkke 87 273257 2009 541981 n/a Intron 4 TACAGAGAATCACCCC eekddddddddddkke 82 273651 2010 541982 n/a Intron 4 GTCCAAGTAAGGTGCT eekddddddddddkke 57 273947 2011 541983 n/a Intron 5 GACCTTGCAGGCTTCC eekddddddddddkke 87 274244 2012 541984 n/a Intron 5 GGGCAAAGGATCCTCT eekddddddddddkke 71 274758 2013 541985 n/a Intron 5 CCCATTCTGCTATCCC eekddddddddddkke 92 275198 2014 541986 n/a Intron 5 GCTGACTAGGAGGGCT eekddddddddddkke 62 275732 2015 541987 n/a Intron 5 CCTGTGAGGTAGTACC eekddddddddddkke 83 276309 2016 541988 n/a Intron 5 GTCCCCCTCCAGTCTA eekddddddddddkke 50 276932 2017 541989 n/a Intron 5 GAGGACTCAATTCCTC eekddddddddddkke 0 277149 2018 541990 n/a Intron 5 GACAAGGTCCTTTTGG eekddddddddddkke 43 277391 2019 541991 n/a Intron 5 GCTCTTGTGTGCACCC eekddddddddddkke 90 277730 2020 541992 n/a Intron 5 TCACCGCCTGCACCAC eekddddddddddkke 75 278342 2021 541993 n/a Intron 5 GGTTGCACTGTGCAAT eekddddddddddkke 26 278917 2022 541994 n/a Intron 6 TTCCACAGGCCTCCAT eekddddddddddkke 64 279303 2023 541995 n/a Intron 6 GCTGAGTTCCATATGC eekddddddddddkke 72 279679 2024 541996 n/a Intron 6 GAACCGCCACCTCAGG eekddddddddddkke 38 280157 2025 541997 n/a Intron 6 GCTCACGGTTGGAGAC eekddddddddddkke 42 280799 2026 541998 n/a Intron 6 TGGGCTCCCATGTTCA eekddddddddddkke 45 281595 2027 541999 n/a Intron 6 TCACTCTACCAACCTC eekddddddddddkke 33 282572 2028 542000 n/a Intron 6 TCCTTGCTTACAGATG eekddddddddddkke 33 283079 2029 542001 n/a Intron 6 TGATGCTAGCATTACC eekddddddddddkke 37 283653 2030 542002 n/a Intron 6 TGGGTAACTGGCTAGT eekddddddddddkke 47 285711 2031 542003 n/a Intron 6 AACCATTCCTCACCAA eekddddddddddkke 53 287181 2032 542004 n/a Intron 6 GCCCTGAACAGTTGAT eekddddddddddkke 37 287895 2033 542005 n/a Intron 6 GGCTCCTATCATACCT eekddddddddddkke 38 288943 2034 542006 n/a Intron 6 TAGGTCTCACAACCCT eekddddddddddkke 10 289638 2035 542007 n/a Intron 6 GTGCATTAGTCTTCCA eekddddddddddkke 74 290035 2036 542008 n/a Intron 7 CAAAAGCCAGGTTAGC eekddddddddddkke 13 290503 2037 542009 n/a Intron 7 CTGCTGTTGACTACCT eekddddddddddkke 50 290924 2038 542010 n/a Intron 7 GTACCTGCCAGCTACT eekddddddddddkke 35 291807 2039 542011 n/a Exon 8- CCTACCTTTGCTGTTT eekddddddddddkke 12 292611 2040 intron 8 junction 542012 n/a Intron 8 AGTCACCAGCCTAAGC eekddddddddddkke 47 292860 2041 542013 n/a Intron 8 AGGCAACCTGGGAGTG eekddddddddddkke 52 293377 2042 542014 n/a Intron 8 TGGCCTTCACAATGGC eekddddddddddkke 33 294052 2043 542015 n/a Intron 8 GGTGAAGTGGGTTGGA eekddddddddddkke 27 294536 2044 542016 n/a Intron 8 GCTGGTTGTCTGCTGC eekddddddddddkke 60 294931 2045 542017 n/a Intron 8 AGTTTGTGACCCCTGC eekddddddddddkke 81 295475 2046 542018 n/a Intron 8 CCACTCAGTGTGAATG eekddddddddddkke 85 295955 2047 542019 n/a Intron 8 CTGGCCTCAGGGCAAT eekddddddddddkke 51 296186 2048 542020 n/a Intron 8 GTAGACTTGGGTAGGT eekddddddddddkke 53 296680 2049 542022 n/a 3′UTR TGGTGCTAAGCTCTCC eekddddddddddkke 67 301009 2050 542023 n/a 3′UTR CATGCTCAAGCTGGAA eekddddddddddkke 47 301280 2051 542024 206 Exon 2 AAGGTCAACAGCAGCT eekddddddddddkke 93 144990 2052 542025 207 Exon 2 CAAGGTCAACAGCAGC eekddddddddddkke 85 144991 2053 542026 208 Exon 2 CCAAGGTCAACAGCAG eekddddddddddkke 82 144992 2054 542027 209 Exon 2 GCCAAGGTCAACAGCA eekddddddddddkke 84 144993 2055

TABLE 183 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 SEQ SEQ ID ID NO: NO: 2 ISIS 1 Start Target % Start SEQ NO Site Region Sequence Chemistry inhibition Site ID NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 86 156891 1370 542034 870 Exon 7 TCTCACACGCACTTCA eekddddddddddkke 49 290368 2056 542035 871 Exon 7 ATCTCACACGCACTTC eekddddddddddkke 39 290369 2057 542036 872 Exon 7 GATCTCACACGCACTT eekddddddddddkke 50 290370 2058 542049 n/a Intron 1 CTTTCATGAATCAAGC eekddddddddddkke 85 17928 2059 542050 n/a Intron 1 TCTTTCATGAATCAAG eekddddddddddkke 54 17929 2060 542051 n/a Intron 1 GTCTTTCATGAATCAA eekddddddddddkke 96 17930 2061 542052 n/a Intron 1 GGTCTTTCATGAATCA eekddddddddddkke 98 17931 2062 542053 n/a Intron 1 ATGGTCTTTCATGAAT eekddddddddddkke 94 17933 2063 542054 n/a Intron 1 GATGGTCTTTCATGAA eekddddddddddkke 73 17934 2064 542055 n/a Intron 1 TGATGGTCTTTCATGA eekddddddddddkke 83 17935 2065 542056 n/a Intron 1 TATATCAATATTCTCC eekddddddddddkke 75 21821 2066 542057 n/a Intron 1 TTATATCAATATTCTC eekddddddddddkke 23 21822 2067 542058 n/a Intron 1 GTTATATCAATATTCT eekddddddddddkke 87 21823 2068 542102 n/a Intron 2 GCCATCACCAAACACC eekddddddddddkke 97 150973 2112 542103 n/a Intron 2 TGCCATCACCAAACAC eekddddddddddkke 90 150974 2113 542104 n/a Intron 2 TTGCCATCACCAAACA eekddddddddddkke 89 150975 2114 542105 n/a Intron 2 TGGTGACTCTGCCTGA eekddddddddddkke 98 151388 2115 542106 n/a Intron 2 CTGGTGACTCTGCCTG eekddddddddddkke 96 151389 2116 542107 n/a Intron 2 GCTGGTGACTCTGCCT eekddddddddddkke 98 151390 2117 542108 n/a Intron 2 TGCTGGTGACTCTGCC eekddddddddddkke 97 151391 2118 542109 n/a Intron 2 CTGCTGGTGACTCTGC eekddddddddddkke 93 151392 2119

TABLE 184 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 NO: 2 ISIS Start Stop Target % SEQ NO Site Site Region Sequence Chemistry inhibition ID NO 541262 156891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 95 1370 542110 153002 153017 Intron 2 AGTAGTCAATATTATT eekddddddddddkke 74 2120 542111 153003 153018 Intron 2 CAGTAGTCAATATTAT eekddddddddddkke 55 2121 542112 153004 153019 Intron 2 CCAGTAGTCAATATTA eekddddddddddkke 97 2122 542113 153922 153937 Intron 2 CCTTTGGGTGAATAGC eekddddddddddkke 90 2123 542114 153923 153938 Intron 2 ACCTTTGGGTGAATAG eekddddddddddkke 71 2124 542115 153924 153939 Intron 2 CACCTTTGGGTGAATA eekddddddddddkke 78 2125 542116 155595 155610 Intron 2 CAACTTGAGGACAATA eekddddddddddkke 89 2126 542118 155597 155612 Intron 2 CTCAACTTGAGGACAA eekddddddddddkke 98 2127 542119 156395 156410 Intron 2 CAGGAAGAAAGGAACC eekddddddddddkke 95 2128 542120 156396 156411 Intron 2 CCAGGAAGAAAGGAAC eekddddddddddkke 83 2129 542121 156397 156412 Intron 2 ACCAGGAAGAAAGGAA eekddddddddddkke 90 2130 542122 156595 156610 Intron 2 TGCAGTCATGTACACA eekddddddddddkke 97 2131 542123 156596 156611 Intron 2 CTGCAGTCATGTACAC eekddddddddddkke 90 2132 542124 156597 156612 Intron 2 TCTGCAGTCATGTACA eekddddddddddkke 81 2133 542125 156890 156905 Intron 2 TGGTTTGTCAATCCTT eekddddddddddkke 97 2134 542126 156892 156907 Intron 2 CTTGGTTTGTCAATCC eekddddddddddkke 99 2135 542127 157204 157219 Intron 2 GCTACAATGCACAGGA eekddddddddddkke 98 2136 542128 157205 157220 Intron 2 TGCTACAATGCACAGG eekddddddddddkke 98 2137 542129 158008 158023 Intron 2 GATATTTATTGCTGTA eekddddddddddkke 61 2138 542130 158009 158024 Intron 2 TGATATTTATTGCTGT eekddddddddddkke 41 2139 542131 158010 158025 Intron 2 CTGATATTTATTGCTG eekddddddddddkke 86 2140 542132 162752 162767 Intron 2 AGGGTCTTTACAAAGT eekddddddddddkke 69 2141 542133 162753 162768 Intron 2 CAGGGTCTTTACAAAG eekddddddddddkke 71 2142 542134 162754 162769 Intron 2 CCAGGGTCTTTACAAA eekddddddddddkke 93 2143 542135 166353 166368 Intron 2 TTCTGCAGTATCCTAG eekddddddddddkke 84 2144 542136 166354 166369 Intron 2 TTTCTGCAGTATCCTA eekddddddddddkke 88 2145 542137 166355 166370 Intron 2 GTTTCTGCAGTATCCT eekddddddddddkke 95 2146 542138 166356 166371 Intron 2 AGTTTCTGCAGTATCC eekddddddddddkke 92 2147 542139 166357 166372 Intron 2 CAGTTTCTGCAGTATC eekddddddddddkke 93 2148 542140 172747 172762 Intron 2 CAAATTCCAGTCCTAG eekddddddddddkke 73 2149 542141 172748 172763 Intron 2 CCAAATTCCAGTCCTA eekddddddddddkke 91 2150 542142 172749 172764 Intron 2 TCCAAATTCCAGTCCT eekddddddddddkke 90 2151 542143 175372 175387 Intron 2 ACCCATTTCATCCATT eekddddddddddkke 94 2152 542144 175373 175388 Intron 2 AACCCATTTCATCCAT eekddddddddddkke 93 2153 542145 175374 175389 Intron 2 GAACCCATTTCATCCA eekddddddddddkke 97 2154 542146 175375 175390 Intron 2 GGAACCCATTTCATCC eekddddddddddkke 96 2155 542147 175376 175391 Intron 2 AGGAACCCATTTCATC eekddddddddddkke 68 2156 542148 189120 189135 Intron 2 GCTTCATGTCTTTCTA eekddddddddddkke 90 2157 542149 189121 189136 Intron 2 TGCTTCATGTCTTTCT eekddddddddddkke 96 2158 542150 189122 189137 Intron 2 GTGCTTCATGTCTTTC eekddddddddddkke 97 2159 542151 189485 189500 Intron 2 TGAGCTTAGCAGTCAC eekddddddddddkke 92 2160 542152 189486 189501 Intron 2 ATGAGCTTAGCAGTCA eekddddddddddkke 95 2161 542153 189487 189502 Intron 2 CATGAGCTTAGCAGTC eekddddddddddkke 95 2162 542154 191143 191158 Intron 2 TACAGACATAGCTCTA eekddddddddddkke 91 2163 542155 191144 191159 Intron 2 ATACAGACATAGCTCT eekddddddddddkke 74 2164 542156 191145 191160 Intron 2 GATACAGACATAGCTC eekddddddddddkke 91 2165 542157 191146 191161 Intron 2 GGATACAGACATAGCT eekddddddddddkke 94 2166 542158 198149 198164 Intron 2 TGTGGCTTTAATTCAC eekddddddddddkke 71 2167 542159 198150 198165 Intron 2 ATGTGGCTTTAATTCA eekddddddddddkke 81 2168 542160 198151 198166 Intron 2 TATGTGGCTTTAATTC eekddddddddddkke 78 2169 542161 199817 199832 Intron 2 TGTTCAGTTGCATCAC eekddddddddddkke 91 2170 542162 199818 199833 Intron 2 GTGTTCAGTTGCATCA eekddddddddddkke 89 2171 542163 199819 199834 Intron 2 TGTGTTCAGTTGCATC eekddddddddddkke 90 2172 542164 210562 210577 Intron 3 CATCTGGATGTGAGGC eekddddddddddkke 90 2173 542165 210563 210578 Intron 3 ACATCTGGATGTGAGG eekddddddddddkke 78 2174 542166 210564 210579 Intron 3 CACATCTGGATGTGAG eekddddddddddkke 55 2175 542167 219020 219035 Intron 3 TCAGGTAATTTCTGGA eekddddddddddkke 82 2176 542168 219021 219036 Intron 3 CTCAGGTAATTTCTGG eekddddddddddkke 73 2177 542169 219022 219037 Intron 3 TCTCAGGTAATTTCTG eekddddddddddkke 40 2178 542170 225568 225583 Intron 3 TGCTTATTTACCTGGG eekddddddddddkke 90 2179 542171 225569 225584 Intron 3 TTGCTTATTTACCTGG eekddddddddddkke 90 2180 542172 225570 225585 Intron 3 TTTGCTTATTTACCTG eekddddddddddkke 79 2181 542173 225571 225586 Intron 3 TTTTGCTTATTTACCT eekddddddddddkke 32 2182 542174 229619 229634 Intron 3 ATGATGTTACTACTAC eekddddddddddkke 63 2183 542175 229620 229635 Intron 3 AATGATGTTACTACTA eekddddddddddkke 53 2184 542176 229621 229636 Intron 3 CAATGATGTTACTACT eekddddddddddkke 12 2185 542177 232827 232842 Intron 3 CCCCTAGAGCAATGGT eekddddddddddkke 76 2186 542178 232828 232843 Intron 3 CCCCCTAGAGCAATGG eekddddddddddkke 83 2187 542179 232829 232844 Intron 3 TCCCCCTAGAGCAATG eekddddddddddkke 49 2188 542180 237676 237691 Intron 3 TCAATTGCAGATGCTC eekddddddddddkke 88 2189 542181 237677 237692 Intron 3 CTCAATTGCAGATGCT eekddddddddddkke 90 2190 542182 237678 237693 Intron 3 GCTCAATTGCAGATGC eekddddddddddkke 81 2191 542183 237679 237694 Intron 3 AGCTCAATTGCAGATG eekddddddddddkke 85 2192 542184 248232 248247 Intron 3 GTATATTCAGTCCAAG eekddddddddddkke 90 2193 542185 248233 248248 Intron 3 AGTATATTCAGTCCAA eekddddddddddkke 94 2194 542186 248234 248249 Intron 3 CAGTATATTCAGTCCA eekddddddddddkke 97 2195

TABLE 185 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 SEQ ID NO: 1 SEQ ID ISIS Start % NO: 2 SEQ ID NO Site Target Region Sequence Chemistry inhibition Start Site NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 93 156891 1370 545316 168 exon 1-intron 1 ACCTCCGAGCTTCGCC eekddddddddddkke 80 3044 2196 junction 545317 173 exon-exon GTAGGACCTCCGAGCT eekddddddddddkke 74 n/a 2197 junction 545318 177 exon-exon ACCTGTAGGACCTCCG eekddddddddddkke 70 n/a 2198 junction 545321 213 Exon 2 CAGTGCCAAGGTCAAC eekddddddddddkke 77 144997 2199 545322 225 Exon 2 ACTTGATCCTGCCAGT eekddddddddddkke 36 145009 2200 545332 361 Exon 4/Intron 3 CTCGCTCAGGTGAACG eekddddddddddkke 57 268024 2201 545333 366 Exon 4/Intron 3 AGTCTCTCGCTCAGGT eekddddddddddkke 88 268029 2202 545337 444 Exon 4-intron 4 CCTTCTGGTATAGAAC eekddddddddddkke 21 268107 2203 junction 545340 570 Exon 5 GCTAGTTAGCTTGATA eekddddddddddkke 39 274130 2204 545343 626 exon 3-exon 4 TCTGGTTGCACTATTT eekddddddddddkke 34 n/a 2205 junction 545344 629 exon 3-exon 4 GGATCTGGTTGCACTA eekddddddddddkke 30 n/a 2206 junction 545345 632 Exon 6 GGTGGATCTGGTTGCA eekddddddddddkke 18 278926 2207 545346 638 Exon 6 GCAATGGGTGGATCTG eekddddddddddkke 50 278932 2208 545347 647 Exon 6 CAGTTGAGGGCAATGG eekddddddddddkke 71 278941 2209 545348 651 Exon 6 AGTCCAGTTGAGGGCA eekddddddddddkke 58 278945 2210 545349 655 Exon 6 GTAAAGTCCAGTTGAG eekddddddddddkke 34 278949 2211 545350 660 Exon 6 GTTCAGTAAAGTCCAG eekddddddddddkke 52 278954 2212 545351 685 Exon 6 CTGCATGAATCCCAGT eekddddddddddkke 77 278979 2213 545355 923 Exon 7 ACATAGAGCACCTCAC eekddddddddddkke 38 290421 2214 545356 926 Exon 7 GTTACATAGAGCACCT eekddddddddddkke 79 290424 2215 545357 929 Exon 7 AGTGTTACATAGAGCA eekddddddddddkke 70 290427 2216 545362 1124 Exon 7-exon 8 TCCTTGAGGAGATCTG eekddddddddddkke 3 n/a 2217 junction 545363 1170 Exon 10 GCTATCATGAATGGCT eekddddddddddkke 69 297587 2218 545364 1180 Exon 10 CGGGTTTATAGCTATC eekddddddddddkke 58 297597 2219 545369 1320 Exon 10 ATCCTTCACCCCTAGG eekddddddddddkke 46 297737 2220 545370 1328 Exon 10 GAGTCGCCATCCTTCA eekddddddddddkke 60 297745 2221 545371 1332 Exon 10 TCCAGAGTCGCCATCC eekddddddddddkke 51 297749 2222 545373 1418 Exon 10 GGCTGAGCAACCTCTG eekddddddddddkke 80 297835 2223 545374 1422 Exon 10 CTGTGGCTGAGCAACC eekddddddddddkke 63 297839 2224 545380 1524 Exon 10 GATAACACTGGGCTGC eekddddddddddkke 60 297941 2225 545381 1530 Exon 10 TGCTTGGATAACACTG eekddddddddddkke 76 297947 2226 545382 1533 Exon 10 CTCTGCTTGGATAACA eekddddddddddkke 60 297950 2227 545386 1600 Exon 10 GCTGAATATGGGCAGC eekddddddddddkke 29 298017 2228 545387 1613 Exon 10 CTTGGATTGCTTAGCT eekddddddddddkke 59 298030 2229 545388 1645 Exon 10 CCTGGGCATAAAAGTC eekddddddddddkke 47 298062 2230 545392 1832 Exon 10 ACCTTGATGTGAGGAG eekddddddddddkke 44 298249 2231

TABLE 186 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 SEQ SEQ ID ID NO: 1 NO: 2 ISIS Start Target % Start NO Site Region Sequence Chemistry inhibition Site SEQ ID NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 89 156891 1370 545393 1838 Exon 10 GATTCAACCTTGATGT eekddddddddddkke 40 298255 2232 545394 1844 Exon 10 ATGTGTGATTCAACCT eekddddddddddkke 80 298261 2233 545395 1956 Exon 10 TGGGACAGGCATCTCA eekddddddddddkke 29 298373 2234 545396 1961 Exon 10 TAGTCTGGGACAGGCA eekddddddddddkke 48 298378 2235 545397 1968 Exon 10 GGAGGTATAGTCTGGG eekddddddddddkke 61 298385 2236 545398 1986 Exon 10 GGACTGTACTATATGA eekddddddddddkke 48 298403 2237 545401 2077 Exon 10 TCAGTTGGTCTGTGCT eekddddddddddkke 60 298494 2238 545402 2095 Exon 10 GCTAAGGCATGATTTT eekddddddddddkke 53 298512 2239 545406 2665 Exon 10 GCCATGCTTGAAGTCT eekddddddddddkke 87 299082 2240 545407 2668 Exon 10 ATAGCCATGCTTGAAG eekddddddddddkke 70 299085 2241 545408 2692 Exon 10 ACACAGTGTGTAGTGT eekddddddddddkke 60 299109 2242 545409 2699 Exon 10 CTGCAGTACACAGTGT eekddddddddddkke 31 299116 2243 545410 2704 Exon 10 ACCAACTGCAGTACAC eekddddddddddkke 57 299121 2244 545411 2739 Exon 10 TAGACTGTAGTTGCTA eekddddddddddkke 53 299156 2245 545412 2747 Exon 10 ACCAGCTTTAGACTGT eekddddddddddkke 56 299164 2246 545413 2945 Exon 10 GTAAGTTGATCTGTGC eekddddddddddkke 79 299362 2247 545414 2963 Exon 10 TACTTCTTTTGGTGCC eekddddddddddkke 82 299380 2248 545416 3212 Exon 10 TCTTGTACCTTATTCC eekddddddddddkke 73 299629 2249 545417 3306 Exon 10 TGGTTATAGGCTGTGA eekddddddddddkke 90 299723 2250 545418 3309 Exon 10 GTCTGGTTATAGGCTG eekddddddddddkke 88 299726 2251 545419 3313 Exon 10 ATGTGTCTGGTTATAG eekddddddddddkke 68 299730 2252 545420 3317 Exon 10 GAGTATGTGTCTGGTT eekddddddddddkke 84 299734 2253 545421 4049 Exon 10 GGTCTGCGATAAATGG eekddddddddddkke 69 300466 2254 545429 4424 Exon 10 GCCAGACACAACTAGT eekddddddddddkke 59 300841 2255 545430 31 Exon 1 ACCGCCACTGTAGCAG eekddddddddddkke 76 2907 2256 545431 36 Exon 1 CCGCCACCGCCACTGT eekddddddddddkke 94 2912 2257 545432 103 Exon 1 GGGCCTCCGGCCCGCG eekddddddddddkke 22 2979 2258 545433 143 Exon 1 AGAGCGCGGGTTCGCG eekddddddddddkke 61 3019 2259 545434 n/a Intron TACTGACCCCAGTTCC eekddddddddddkke 68 3654 2260 1/Exon 1 545435 n/a Intron ACTCTACTGACCCCAG eekddddddddddkke 70 3658 2261 1/Exon 1 545436 n/a Intron GTCACTCTACTGACCC eekddddddddddkke 83 3661 2262 1/Exon 1 545437 n/a Intron TTCATGCGGACTGGTG eekddddddddddkke 68 3680 2263 1/Exon 1 545438 n/a Intron GTGAGCATGGACCCCA eekddddddddddkke 94 225436 2264 3/Exon 3 545439 n/a Intron TGATATGTGAGCATGG eekddddddddddkke 88 225442 2265 3/Exon 3 545440 n/a Intron AAGTTGGTGAGCTTCT eekddddddddddkke 85 226785 2266 3/Exon 3 545441 n/a Intron CCTTCAAGTTGGTGAG eekddddddddddkke 88 226790 2267 3/Exon 3 545442 n/a Intron GTAAGATCCTTTTGCC eekddddddddddkke 70 226883 2268 3/Exon 3 545443 n/a Intron CAGCTGTGCAACTTGC eekddddddddddkke 50 238345 2269 3/Exon 3 545444 n/a Intron GCCTTGGTAGGTAGGG eekddddddddddkke 68 238422 2270 3/Exon 3 545445 n/a Intron AGAGCCTTGGTAGGTA eekddddddddddkke 85 238425 2271 3/Exon 3 545446 n/a Intron CCCGCACAAACGCGCA eekddddddddddkke 10 3614 2272 1/Exon 1 545447 n/a Intron GTCTTCAAGGTCAGTT eekddddddddddkke 92 93208 2273 1/Exon 1 545448 n/a Intron GCCCAGTGAATTCAGC eekddddddddddkke 76 93246 2274 1/Exon 1 545449 n/a Intron AGATGCGCCCAGTGAA eekddddddddddkke 60 93252 2275 1/Exon 1 545450 n/a Intron GTAAGATGCGCCCAGT eekddddddddddkke 78 93255 2276 1/Exon 1 545451 n/a Intron CCAGAAGGCACTTGTA eekddddddddddkke 42 93301 2277 1/Exon 1 545452 n/a Intron GGAAGATTTGCAGAAC eekddddddddddkke 15 93340 2278 1/Exon 1 545453 n/a Intron CCTTGGTCATGGAAGA eekddddddddddkke 35 93350 2279 1/Exon 1 545454 n/a Intron TGACCTTGGTCATGGA eekddddddddddkke 55 93353 2280 1/Exon 1 545455 n/a Intron GAGGTGACCTTGGTCA eekddddddddddkke 70 93357 2281 1/Exon 1 545456 n/a Intron ATCCAAAGAGGTGACC eekddddddddddkke 41 93364 2282 1/Exon 1 545457 n/a Intron GCCAATCCAAAGAGGT eekddddddddddkke 56 93368 2283 1/Exon 1 545458 n/a Intron GGTCTGCCAATCCAAA eekddddddddddkke 79 93373 2284 1/Exon 1 545459 n/a Intron CCCTGGGTCTGCCAAT eekddddddddddkke 68 93378 2285 1/Exon 1 545460 n/a Intron GAGATCTCAACAAGGG eekddddddddddkke 52 93427 2286 1/Exon 1 545461 n/a Intron CGCCCATCACTCTTCC eekddddddddddkke 68 93988 2287 1/Exon 1 545462 n/a Intron CACCTGTCGCCCATCA eekddddddddddkke 67 93995 2288 1/Exon 1 545463 n/a Intron CATCACCTGTCGCCCA eekddddddddddkke 78 93998 2289 1/Exon 1 545464 n/a Intron CACCATCACCTGTCGC eekddddddddddkke 74 94001 2290 1/Exon 1 545465 n/a Intron AATAGTTGTCACCATC eekddddddddddkke 76 94010 2291 1/Exon 1 545466 n/a Intron GCCACCTTTCATGAGA eekddddddddddkke 58 94048 2292 1/Exon 1 545467 n/a Intron CTCTTGGAAGTAGGTA eekddddddddddkke 89 198762 2293 2/Exon 2 545468 n/a Intron GTTCTCTTGGAAGTAG eekddddddddddkke 80 198765 2294 2/Exon 2 545469 n/a Intron TAAACAGGTTGGTCTG eekddddddddddkke 68 198854 2295 2/Exon 2

Example 121 Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by Deoxy, MOE and (S)-cEt Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 187 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541396 30 51 68 74 67 1.4 541262 55 87 90 94 97 0.2 541393 30 38 52 66 81 2.1 541375 41 45 54 64 79 1.6 541438 44 49 75 80 91 0.9 541428 35 32 56 78 88 1.8 541491 13 46 67 55 95 2.0 541435 21 46 55 72 94 1.9 541471 11 49 50 77 89 2.0 541430 24 44 56 57 79 2.2 541492 32 40 65 80 85 1.5 541431 22 46 73 84 92 1.5

TABLE 188 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541487 36 46 66 85 92 1.3 541423 33 55 64 80 93 1.2 541452 37 60 79 87 94 0.9 541505 51 75 86 92 97 0.4 541522 54 76 81 90 95 0.3 541539 65 76 85 94 98 0.2 541503 54 65 80 93 97 0.5 541520 43 61 86 94 96 0.7 541515 57 72 85 92 94 0.3 541564 57 72 88 90 97 0.3 541554 43 65 81 89 93 0.7 541509 11 8 19 6 8 >10 541584 59 65 84 91 96 0.3 541585 70 80 93 92 98 0.1

TABLE 189 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541598 26 43 75 80 76 1.5 541592 35 48 67 85 95 1.2 541641 22 63 70 91 93 1.2 541590 27 59 70 94 95 1.2 541615 40 65 84 88 94 0.7 541595 35 57 73 84 95 1.0 541575 49 60 79 84 95 0.6 541571 41 50 76 80 94 1.0 541582 0 10 25 50 82 4.4 541262 66 79 93 94 99 <0.6 541652 1 44 80 82 87 1.9 541670 29 40 63 79 89 1.6 541662 17 13 45 62 84 3.1 541724 37 47 72 85 95 1.2

TABLE 190 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541748 86 94 96 98 98 <0.6 541767 83 91 95 96 98 <0.6 541797 78 89 93 97 99 <0.6 541766 59 82 92 97 99 <0.6 541742 65 87 93 95 99 <0.6 541750 80 86 96 96 99 <0.6 541262 79 88 93 97 97 <0.6 541749 71 84 93 95 98 <0.6 541793 71 88 94 97 98 <0.6 541785 56 79 89 93 98 <0.6 541746 34 61 85 94 97 0.9 541752 49 72 88 93 93 <0.6 541826 86 94 95 99 98 <0.6 541811 66 87 93 97 98 <0.6

TABLE 191 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541822 83 88 95 96 96 <0.6 541870 77 87 95 97 98 <0.6 541262 85 93 96 97 98 <0.6 541873 32 77 93 94 97 0.7 541819 60 91 97 97 99 <0.6 541841 86 91 95 96 97 <0.6 541825 78 88 95 98 98 <0.6 541863 63 77 87 93 97 <0.6 541827 42 80 87 94 97 <0.6 541875 77 84 93 96 97 <0.6 541835 56 73 90 95 98 <0.6 541838 72 90 93 98 97 <0.6 541833 52 69 83 92 97 <0.6 541813 47 75 86 95 97 <0.6

TABLE 192 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541853 74 79 88 93 91 <0.6 541842 69 85 91 97 99 <0.6 541877 79 91 93 98 97 <0.6 541848 58 90 96 98 98 0.7 541804 23 81 89 95 95 0.8 541881 87 94 98 98 99 <0.6 541936 91 96 98 99 98 <0.6 541909 56 80 89 95 97 <0.6 541907 75 91 95 97 98 <0.6 541952 68 81 93 97 98 <0.6 541953 68 80 94 97 98 <0.6 541914 60 78 94 97 97 <0.6 541880 56 74 89 94 95 <0.6 541903 37 74 87 96 98 0.6

TABLE 193 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541895 47 72 85 93 94 <0.6 541882 60 67 89 93 97 <0.6 541889 63 80 87 94 97 <0.6 541904 26 78 23 89 93 1.4 545418 0 81 91 94 95 1.7 541930 58 71 82 88 92 <0.6 545439 67 87 93 96 98 <0.6 542024 15 58 78 87 90 1.4 541985 59 81 88 93 97 <0.6 541972 47 58 83 90 92 0.6 541991 57 64 88 92 83 <0.6 541980 33 50 76 72 93 1.2

TABLE 194 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541264 26 44 64 79 89 1.6 541265 29 32 62 79 91 1.8 541263 25 40 62 78 93 1.7 541268 57 73 85 90 95 0.3 541266 15 33 46 66 90 2.5 542107 93 97 98 98 98 <0.6 542052 93 96 97 96 98 <0.6 542105 80 92 96 98 97 <0.6 542102 94 96 96 97 98 <0.6 542108 90 92 94 97 99 <0.6 542080 87 93 95 95 97 <0.6

TABLE 195 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 542101 90 97 97 97 95 <0.6 542051 89 96 95 98 97 <0.6 542106 83 93 96 96 98 <0.6 542071 84 91 94 97 97 <0.6 542094 85 92 94 97 98 <0.6 542069 89 94 97 95 98 <0.6 542086 83 94 96 97 98 <0.6 542085 85 92 96 97 97 <0.6 542053 64 83 94 98 97 <0.6 542087 69 84 99 95 98 <0.6 542109 87 94 96 98 98 <0.6 542126 96 98 99 98 98 <0.6 542127 94 96 97 98 97 <0.6 542128 90 96 98 98 97 <0.6

TABLE 196 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 542118 97 97 98 95 43 <0.6 542186 93 96 98 99 98 <0.6 542150 95 97 98 99 99 <0.6 542122 90 94 98 98 99 <0.6 542125 88 97 98 98 99 <0.6 542145 90 96 98 99 99 <0.6 542112 86 94 99 99 99 <0.6 542149 88 93 99 98 99 <0.6 542146 79 93 96 97 98 <0.6 542153 87 94 97 98 99 <0.6 542119 64 84 93 97 98 <0.6 542137 76 91 97 97 98 <0.6 542152 84 94 96 96 97 <0.6 542157 83 95 98 99 98 <0.6

TABLE 197 0.625 1.250 2.50 5.00 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 542185 82 93 96 96 94 <0.6 542143 81 91 96 98 98 <0.6 542144 77 93 95 96 99 <0.6 542139 87 93 98 98 98 <0.6 542134 83 90 90 95 96 <0.6 545333 68 85 91 96 98 <0.6 545373 57 73 86 92 97 <0.6 545438 84 96 98 97 99 <0.6 545431 77 91 93 97 98 <0.6 545447 70 85 96 96 97 <0.6 545417 62 82 90 93 95 <0.6 545467 77 88 91 94 95 <0.6 545441 63 82 92 94 96 <0.6

Example 122 Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by Deoxy, MOE and (S)-cEt Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.04 μM, 0.11 μM, 0.33 μM, 1.00 μM, and 3.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 198 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 11 16 57 93 98 0.2 541724 0 27 71 66 83 0.3 541748 28 40 71 90 97 0.1 541767 19 38 54 87 98 0.2 541797 23 46 70 88 97 0.1 541766 15 26 49 82 96 0.3 541742 17 28 41 80 95 0.3 541750 33 27 60 89 98 0.2 541749 27 16 62 84 82 0.2 541793 0 14 44 77 96 0.4 541785 4 11 39 75 95 0.4 541752 14 6 45 70 94 0.4 541826 8 34 74 94 99 0.2 541811 6 4 45 79 97 0.4 541822 9 29 67 89 97 0.2

TABLE 199 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 0 16 47 82 98 0.4 541819 3 12 50 76 94 0.3 541841 0 19 47 80 95 0.3 541825 0 6 40 74 96 0.4 541827 5 26 48 76 95 0.3 541835 7 11 33 74 93 0.4 541838 21 26 61 90 97 0.2 541833 0 9 41 63 89 0.5 541813 0 17 28 65 92 0.5 541842 5 15 30 72 90 0.4 541804 0 12 3 49 79 1.1 542024 0 0 26 54 76 1.0 542107 15 45 78 92 99 0.1 542105 2 14 55 88 98 0.3 542102 10 16 73 88 98 0.2

TABLE 200 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 4 18 50 86 95 0.3 542108 15 13 65 86 97 0.2 542101 17 40 68 92 98 0.2 542106 4 23 56 88 98 0.3 542094 0 30 51 86 96 0.3 542086 13 38 50 84 97 0.2 542085 0 27 57 90 98 0.3 542087 7 3 49 80 92 0.4 542109 17 10 56 88 98 0.3 542126 40 63 91 96 99 <0.03 542127 27 47 69 93 97 0.1 542128 11 30 66 90 98 0.2 542118 14 42 77 95 98 0.1 542150 31 46 72 94 98 0.1 542122 13 14 59 90 97 0.3

TABLE 201 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 0 2 50 86 97 0.4 542125 31 32 69 89 96 0.1 542145 15 29 64 91 97 0.2 542112 14 38 61 87 96 0.2 542149 9 37 63 90 97 0.2 542146 13 33 59 82 95 0.2 542153 22 26 63 86 96 0.2 542119 10 20 34 70 87 0.4 542137 3 19 47 77 95 0.3 542152 0 9 47 82 96 0.4 542157 0 26 56 84 96 0.3 542143 8 12 44 81 95 0.3 542144 0 21 42 75 95 0.4 542139 0 14 46 82 97 0.4 542134 3 23 43 72 92 0.4

TABLE 202 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 0 9 64 85 97 0.3 541870 7 15 48 80 92 0.3 541262 0 29 63 90 98 0.2 541863 0 26 40 82 93 0.4 541875 6 30 71 84 91 0.2 541853 0 13 39 67 91 0.5 541877 0 26 41 79 94 0.4 541881 0 30 54 87 94 0.3 541936 20 41 73 93 98 0.1 541909 0 16 34 64 90 0.5 541907 6 31 59 84 96 0.2 541952 0 0 50 72 92 0.5 541953 0 22 50 80 92 0.4 541914 0 0 46 76 93 0.4 541880 0 13 48 79 89 0.4

TABLE 203 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 0 5 53 78 94 0.4 541903 12 20 26 62 88 0.5 541895 3 12 29 66 92 0.5 541882 2 0 27 65 86 0.7 541889 12 12 47 68 87 0.4 541930 0 6 40 59 85 0.6 541985 0 16 41 66 93 0.4 542031 1 0 22 55 80 0.8 541972 0 1 23 46 83 0.9 541991 4 35 42 67 89 0.4 542052 5 28 70 92 98 0.2 542080 0 18 54 87 96 0.3 542051 0 18 52 86 97 0.3 542071 5 3 51 74 95 0.4 542069 0 7 56 85 94 0.3

TABLE 204 0.04 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 539380 11 20 54 89 92 0.3 542053 6 14 38 69 74 0.6 542186 14 43 70 90 98 0.2 542185 0 26 48 80 96 0.3 545333 0 4 27 65 90 0.6 545336 0 15 24 43 79 0.9 545373 0 2 9 42 86 1.0 545438 0 24 56 81 92 0.3 545431 0 18 50 73 91 0.4 545447 0 15 34 78 93 0.4 545417 0 11 39 66 87 0.5 545467 12 16 37 76 93 0.4 545441 21 15 20 60 87 0.6 545439 17 24 49 82 91 0.3

Example 123 Dose-Dependent Antisense Inhibition of Rhesus Monkey GHR in LLC-MK2 Cells

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested for their potency for rhesus GHR mRNA in LLC-MK2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 205 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No Chemistry μM μM μM μM μM (μM) 541262 Deoxy, MOE and 9 25 42 85 91 1.1 (S)-cEt 541742 Deoxy, MOE and 0 24 19 58 77 3.2 (S)-cEt 541767 Deoxy, MOE and 6 10 30 68 88 2.0 (S)-cEt 541875 Deoxy, MOE and 7 19 64 84 96 0.9 (S)-cEt 541881 Deoxy, MOE and 6 24 59 79 91 1.0 (S)-cEt 542101 Deoxy, MOE and 0 5 38 71 81 2.0 (S)-cEt 542112 Deoxy, MOE and 5 17 33 67 76 2.0 (S)-cEt 542118 Deoxy, MOE and 1 6 35 68 86 2.0 (S)-cEt 542125 Deoxy, MOE and 0 12 57 83 93 1.0 (S)-cEt 542127 Deoxy, MOE and 1 0 30 68 84 2.4 (S)-cEt 542128 Deoxy, MOE and 12 0 26 58 83 2.7 (S)-cEt 542153 Deoxy, MOE and 4 0 0 36 59 6.6 (S)-cEt 542185 Deoxy, MOE and 4 0 25 56 87 2.5 (S)-cEt 542186 Deoxy, MOE and 15 23 51 73 90 1.1 (S)-cEt 542051 Deoxy, MOE and 5 19 40 81 94 1.2 (S)-cEt

TABLE 206 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No Chemistry μM μM μM μM μM (μM) 523723 5-10-5 MOE 23 14 31 43 71 3.5 532254 5-10-5 MOE 29 35 42 69 87 0.8 532401 5-10-5 MOE 27 28 46 73 88 1.2 533932 5-10-5 MOE 10 24 48 70 92 1.2 539376 3-10-4 MOE 21 8 8 35 81 4.3 539399 3-10-4 MOE 2 10 14 18 57 8.3 539404 3-10-4 MOE 39 12 25 27 57 7.7 539416 3-10-4 MOE 24 35 44 79 89 1.0 539432 3-10-4 MOE 9 29 42 73 89 1.2 541262 Deoxy, MOE 0 43 63 88 94 0.8 and (S)-cEt 541742 Deoxy, MOE 3 19 35 56 85 1.9 and (S)-cEt 541767 Deoxy, MOE 3 24 39 64 86 1.6 and (S)-cEt 545439 Deoxy, MOE 19 15 43 74 80 1.7 and (S)-cEt 545447 Deoxy, MOE 25 34 58 80 90 0.6 and (S)-cEt

Example 124 Dose-Dependent Antisense Inhibition of GHR in Cynomolgus Primary Hepatocytes

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested for their potency for GHR mRNA in cynomolgus monkey primary hepatocytes. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 207 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No Chemistry μM μM μM μM μM (μM) 541262 Deoxy, MOE 40 52 75 92 98 0.3 and (S)-cEt 541742 Deoxy, MOE 40 57 51 91 96 0.2 and (S)-cEt 541767 Deoxy, MOE 36 59 60 78 91 0.4 and (S)-cEt 541875 Deoxy, MOE 54 76 88 95 95 <0.1 and (S)-cEt 541881 Deoxy, MOE 53 75 85 98 98 <0.1 and (S)-cEt 542101 Deoxy, MOE 38 55 78 89 97 0.2 and (S)-cEt 542112 Deoxy, MOE 28 50 74 89 96 0.4 and (S)-cEt 542118 Deoxy, MOE 20 45 69 84 91 0.5 and (S)-cEt 542125 Deoxy, MOE 33 62 77 92 97 0.3 and (S)-cEt 542127 Deoxy, MOE 30 50 65 86 92 0.4 and (S)-cEt 542128 Deoxy, MOE 25 40 52 80 93 0.7 and (S)-cEt 542153 Deoxy, MOE 10 31 51 73 85 1.0 and (S)-cEt 542185 Deoxy, MOE 12 45 65 85 93 0.6 and (S)-cEt 542186 Deoxy, MOE 36 54 74 90 96 0.3 and (S)-cEt 542051 Deoxy, MOE 9 29 32 32 42 >10 and (S)-cEt

TABLE 208 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No Chemistry μM μM μM μM μM (μM) 523435 5-10-5 35 47 61 74 85 0.5 MOE 523723 5-10-5 4 16 40 66 86 1.8 MOE 532254 5-10-5 14 15 24 16 9 >10 MOE 532401 5-10-5 37 54 73 88 94 0.3 MOE 533932 5-10-5 23 40 69 78 86 0.6 MOE 539376 3-10-4 3 0 44 65 91 2.0 MOE 539399 3-10-4 0 0 9 42 67 5.0 MOE 539404 3-10-4 0 0 26 52 71 3.5 MOE 539416 3-10-4 8 29 62 89 93 0.7 MOE 539432 3-10-4 0 24 55 85 93 0.9 MOE 541262 Deoxy, 23 52 73 92 96 0.4 MOE and (S)-cEt 541742 Deoxy, 15 51 73 86 97 0.5 MOE and (S)-cEt 541767 Deoxy, 19 20 39 68 81 1.8 MOE and (S)-cEt 545439 Deoxy, 0 0 30 61 90 2.4 MOE and (S)-cEt 545447 Deoxy, 0 17 17 19 27 >10 MOE and (S)-cEt

Example 125 Dose-Dependent Antisense Inhibition of GHR in Hep3B Cells

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested for their potency for GHR mRNA at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 209 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 541262 25 43 76 85 94 0.5 541742 32 55 76 88 97 0.3 541767 29 56 83 89 97 0.3 541875 38 68 84 93 94 0.1 541881 32 57 81 94 97 0.3 542051 34 66 83 95 98 0.2 542101 25 55 85 95 98 0.3 542112 18 56 83 95 98 0.4 542118 42 61 88 95 97 0.1 542125 30 63 87 95 98 0.2 542127 50 70 91 91 98 0.1 542128 38 63 88 96 98 0.2 542153 37 59 85 94 97 0.2 542185 44 51 76 89 96 0.2 542186 46 59 84 95 97 0.1

TABLE 210 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 523435 9 26 49 78 93 1.0 523723 7 16 39 72 90 1.4 532254 36 46 69 86 94 0.4 532401 25 54 71 86 91 0.4 533932 8 47 69 80 94 0.7 539376 26 31 54 73 86 0.8 539399 23 43 72 89 94 0.5 539404 30 60 88 95 98 0.2 539416 30 59 84 93 98 0.3 539432 35 62 88 95 98 0.2 541262 43 60 84 89 98 0.2 541742 23 53 73 84 97 0.4 541767 22 49 74 85 92 0.4 545439 41 69 88 95 96 0.1 545447 31 47 63 74 82 0.5

Example 126 Dose-Dependent Antisense Inhibition of GHR in Cynomolgus Primary Hepatocytes

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in cynomolgous monkey primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 0.04 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 211 0.04 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No μM μM μM μM μM μM (μM) 541767 8 17 29 48 59 58 0.4 541875 20 39 48 51 55 58 0.2 541881 23 36 49 60 56 58 0.1 542112 23 21 35 42 54 68 0.5 542118 19 14 26 38 54 59 0.8 542153 17 20 27 39 46 52 2.2 542185 20 23 27 46 39 56 2.0 532254 1 20 23 11 1 23 >10 532401 0 15 24 39 47 55 1.6 523723 0 0 7 24 49 54 2.0

Example 127 Comparative Analysis of Dose-Dependent Antisense Inhibition of GHR in Hep3B Cells

ISIS 532401 was compared with specific antisense oligonucleotides disclosed in U.S. 2006/0178325 by testing at various doses in Hep3B cells. The oligonucleotides were selected based on the potency demonstrated in studies described in the application. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.11 μM, 0.33 μM, 1.00 μM, 1.11 μM, 3.00 μM, and 9.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. The results indicate that ISIS 532401 was markedly more potent than the most potent oligonucleotides of US 2006/0178325.

TABLE 212 0.11 0.33 1.00 3.00 9.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 227452 11 12 46 73 92 1.4 227488 26 25 39 76 88 1.2 272309 16 14 39 66 91 1.6 272322 13 20 44 70 86 1.4 272328 22 20 24 43 56 5.7 272338 22 24 52 71 85 1.1 532401 34 53 72 87 94 0.3

Example 128 Tolerability of 5-10-5 MOE Gapmers Targeting Human GHR in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotides (100 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 213. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 213 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 31 50 0.28 0.15 28 ISIS 523271 366 285 0.18 0.11 29 ISIS 523324 222 139 0.19 0.10 31 ISIS 523604 2106 1157 0.41 0.06 48 ISIS 532254 66 84 0.11 0.10 27 ISIS 533121 176 155 0.19 0.09 27 ISIS 533161 1094 904 0.23 0.07 29 ISIS 533178 78 83 0.18 0.08 28 ISIS 533234 164 147 0.21 0.09 26

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin content. The results are presented in Table 214. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 214 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 45 13 8.2 4.1 689 ISIS 523271 42 12 7.9 4.5 1181 ISIS 523324 39 11 7.5 7.9 980 ISIS 523604 33 10 6.9 14.1 507 ISIS 532254 35 10 6.9 7.2 861 ISIS 533121 39 12 7.9 8.4 853 ISIS 533161 49 14 9.3 9.0 607 ISIS 533178 44 13 8.5 6.9 765 ISIS 533234 42 12 7.8 9.2 1045

Example 129 Tolerability of 5-10-5 MOE Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (100 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 215. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 215 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 30 59 0.26 0.14 20 ISIS 523715 636 505 0.24 0.14 22 ISIS 523723 57 80 0.20 0.16 23 ISIS 523726 165 167 0.18 0.15 23 ISIS 523736 140 177 0.20 0.15 23 ISIS 523747 96 108 0.17 0.14 23 ISIS 523789 45 74 0.20 0.15 22 ISIS 532395 64 111 0.23 0.12 21 ISIS 532401 47 88 0.21 0.17 22 ISIS 532411 225 426 0.17 0.16 22 ISIS 532420 60 99 0.21 0.12 25 ISIS 532468 319 273 0.15 0.14 21 ISIS 533932 62 81 0.18 0.14 21

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WB), RBC, and platelets, and total hemoglobin content. The results are presented in Table 216. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 216 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 43 13 8.1 3.3 1047 ISIS 523715 40 12 8.1 4.2 1153 ISIS 523723 35 11 6.8 2.9 1154 ISIS 523726 32 10 6.8 5.8 1056 ISIS 523736 35 11 7.1 3.6 1019 ISIS 523747 37 11 7.7 2.8 1146 ISIS 523789 37 11 7.3 2.5 1033 ISIS 532395 37 11 7.4 4.5 890 ISIS 532401 36 11 7.1 3.7 1175 ISIS 532411 27 8 5.3 3.2 641 ISIS 532420 35 11 7.0 3.3 1101 ISIS 532468 36 11 7.4 4.0 1043 ISIS 533932 36 11 7.2 3.8 981

Example 130 Tolerability of 3-10-4 MOE Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (100 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 217. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 217 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 48 63 0.20 0.13 28 ISIS 539302 204 192 0.15 0.15 24 ISIS 539321 726 455 0.17 0.12 27 ISIS 539360 3287 2495 0.58 0.13 22 ISIS 539361 310 226 0.17 0.11 21 ISIS 539376 77 75 0.14 0.12 27 ISIS 539379 134 136 0.16 0.13 24 ISIS 539380 180 188 0.14 0.12 23 ISIS 539383 80 81 0.15 0.12 25 ISIS 539399 119 127 0.13 0.12 24 ISIS 539401 1435 1172 0.24 0.11 24 ISIS 539403 1543 883 0.18 0.12 26 ISIS 539404 75 109 0.16 0.13 23 ISIS 539416 100 107 0.19 0.15 26 ISIS 539432 55 64 0.20 0.14 22 ISIS 539433 86 91 0.12 0.13 22

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin content. The results are presented in Table 218. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 218 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 46 13 8.5 6 954 ISIS 539302 40 11 8.1 13 830 ISIS 539321 39 11 7.8 16 723 ISIS 539360 49 14 9.0 14 671 ISIS 539361 45 13 8.5 9 893 ISIS 539376 42 12 7.7 6 988 ISIS 539379 42 12 8.1 7 795 ISIS 539380 38 10 7.7 8 950 ISIS 539383 45 12 8.4 8 795 ISIS 539399 41 12 8.0 10 895 ISIS 539401 41 11 8.2 9 897 ISIS 539403 33 9 6.2 13 1104 ISIS 539404 42 12 8.4 7 641 ISIS 539416 41 11 7.5 5 686 ISIS 539432 44 12 8.0 6 920 ISIS 539433 40 11 7.4 6 987

Example 131 Tolerability of Deoxy, MOE and (S)-cEt Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS oligonucleotide (50 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 219. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 219 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 36 71 0.22 0.18 22 ISIS 541262 115 133 0.21 0.18 21 ISIS 541724 543 531 0.34 0.17 21 ISIS 541742 44 71 0.18 0.16 21 ISIS 541748 269 582 0.16 0.15 22 ISIS 541749 626 491 0.20 0.20 22 ISIS 541750 1531 670 0.20 0.18 23 ISIS 541766 2107 1139 0.21 0.21 23 ISIS 541767 42 62 0.21 0.17 20 ISIS 541822 493 202 0.13 0.16 22 ISIS 541826 889 398 0.21 0.14 17 ISIS 541838 266 172 0.16 0.15 20 ISIS 541870 445 272 0.23 0.16 23 ISIS 541875 103 114 0.20 0.15 20 ISIS 541907 940 725 0.16 0.19 35 ISIS 541991 1690 1733 0.31 0.20 23

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin content. The results are presented in Table 220. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 220 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 37 11 7 3 1083 ISIS 541262 38 11 7 6 1010 ISIS 541724 52 16 10 9 940 ISIS 541742 47 14 9 6 1134 ISIS 541748 41 12 8 7 941 ISIS 541749 41 12 8 5 1142 ISIS 541750 42 12 8 4 1409 ISIS 541766 39 11 7 7 989 ISIS 541767 46 14 9 2 994 ISIS 541822 42 12 8 3 1190 ISIS 541826 41 12 8 10 1069 ISIS 541838 44 13 8 6 1005 ISIS 541870 38 11 7 8 1020 ISIS 541875 44 13 8 6 1104 ISIS 541907 40 11 8 9 1271 ISIS 541991 34 10 6 6 1274

Example 132 Tolerability of Deoxy, MOE and (S)-cEt Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers. The 3-10-4 MOE gapmer ISIS 539376 was also included in the study.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS oligonucleotide (50 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 221. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 221 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 43 66 0.21 0.11 20 ISIS 541881 63 109 0.28 0.13 23 ISIS 541936 3260 2108 0.40 0.13 24 ISIS 542051 97 119 0.23 0.12 23 ISIS 542052 454 236 0.23 0.12 25 ISIS 542069 293 211 0.23 0.13 27 ISIS 542085 91 87 0.18 0.10 21 ISIS 542086 137 133 0.24 0.10 23 ISIS 542094 86 143 0.23 0.13 21 ISIS 542101 46 74 0.19 0.10 21 ISIS 542102 4920 2432 2.30 0.15 29 ISIS 542105 1255 575 0.35 0.13 21 ISIS 542106 3082 2295 3.42 0.17 23 ISIS 542107 4049 3092 0.50 0.14 20 ISIS 542108 1835 859 0.32 0.11 21 ISIS 539376 40 79 0.27 0.08 22

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 222. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 222 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC (%) (g/dL) (10⁶/μL) (10³/μL) PBS 46 13 8 6 ISIS 541881 53 15 10 7 ISIS 541936 41 11 8 18 ISIS 542051 49 14 9 8 ISIS 542052 46 13 9 9 ISIS 542069 43 13 8 7 ISIS 542085 38 11 7 5 ISIS 542086 49 14 9 9 ISIS 542094 36 10 6 5 ISIS 542101 44 13 9 5 ISIS 542102 27 7 5 25 ISIS 542105 42 12 8 7 ISIS 542106 37 10 7 14 ISIS 542107 41 12 7 17 ISIS 542108 51 14 8 10 ISIS 539376 49 14 10 5

Example 133 Tolerability of Deoxy, MOE and (S)-cEt Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS oligonucleotide (50 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 223. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 223 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 51 63 0.3 0.14 27 ISIS 542109 3695 2391 0.8 0.19 24 ISIS 542112 119 104 0.3 0.16 28 ISIS 542118 66 86 0.3 0.15 26 ISIS 542122 1112 350 0.3 0.16 27 ISIS 542125 79 92 0.2 0.13 26 ISIS 542126 381 398 0.5 0.14 23 ISIS 542127 54 85 0.3 0.16 26 ISIS 542128 55 89 0.2 0.12 24 ISIS 542145 834 671 0.3 0.11 24 ISIS 542146 163 107 0.2 0.14 30 ISIS 542149 974 752 0.3 0.12 26 ISIS 542150 2840 2126 2.4 0.17 23 ISIS 542153 53 75 0.2 0.14 28 ISIS 542157 137 122 0.3 0.13 25 ISIS 542185 57 72 0.2 0.11 23 ISIS 542186 62 84 0.2 0.12 24 ISIS 545431 2622 1375 3.0 0.15 28 ISIS 545438 1710 1000 0.3 0.14 26 ISIS 545439 70 117 0.2 0.12 28 ISIS 545447 141 108 0.3 0.13 26

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 224. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 224 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 40 12 7 6 1210 ISIS 542109 47 13 9 16 1244 ISIS 542112 50 13 8 7 1065 ISIS 542118 42 12 8 8 1120 ISIS 542122 37 11 7 7 1064 ISIS 542125 42 13 8 7 1063 ISIS 542126 34 10 7 9 1477 ISIS 542127 41 12 7 7 1144 ISIS 542128 40 12 7 6 1196 ISIS 542145 42 12 8 8 1305 ISIS 542146 45 13 8 7 1310 ISIS 542149 33 10 6 12 903 ISIS 542150 27 7 5 18 1202 ISIS 542153 46 13 8 5 1130 ISIS 542157 44 12 9 6 791 ISIS 542185 45 13 8 3 1031 ISIS 542186 44 12 8 6 985 ISIS 545431 28 7 6 13 2609 ISIS 545438 40 11 8 8 1302 ISIS 545439 48 13 9 4 857 ISIS 545447 45 13 9 9 964

Example 134 Tolerability of MOE Gapmers Targeting Human GHR in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (100 mg/kg weekly dose). Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 225 expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in Table 225 expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 225 Liver function markers in Sprague-Dawley rats ALT AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 69 90 0.15 ISIS 523723 79 123 0.12 ISIS 523789 71 105 0.15 ISIS 532254 67 97 0.14 ISIS 532401 61 77 0.12 ISIS 532420 102 127 0.17 ISIS 533178 157 219 0.34 ISIS 533234 71 90 0.11 ISIS 533932 58 81 0.12 ISIS 539376 75 101 0.14 ISIS 539380 86 128 0.16 ISIS 539383 64 94 0.14 ISIS 539399 52 95 0.14 ISIS 539404 88 118 0.13 ISIS 539416 63 104 0.14 ISIS 539432 63 90 0.13 ISIS 539433 69 92 0.13

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 226, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 226 Kidney function markers (mg/dL) in Sprague-Dawley rats BUN Creatinine PBS 24 0.32 ISIS 523723 20 0.39 ISIS 523789 19 0.37 ISIS 532254 21 0.43 ISIS 532401 17 0.36 ISIS 532420 20 0.31 ISIS 533178 20 0.43 ISIS 533234 22 0.41 ISIS 533932 19 0.43 ISIS 539376 19 0.36 ISIS 539380 18 0.35 ISIS 539383 19 0.35 ISIS 539399 18 0.39 ISIS 539404 23 0.39 ISIS 539416 17 0.39 ISIS 539432 20 0.39 ISIS 539433 20 0.34

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 227. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 227 Hematology markers in Sprague-Dawley rats HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 46 15 8 11 1078 ISIS 523723 38 12 7 19 626 ISIS 523789 38 12 8 12 702 ISIS 532254 36 12 7 11 547 ISIS 532401 42 14 8 12 858 ISIS 532420 37 12 7 17 542 ISIS 533178 37 12 7 15 1117 ISIS 533234 38 12 7 8 657 ISIS 533932 40 13 7 9 999 ISIS 539376 43 14 9 8 910 ISIS 539380 33 11 5 6 330 ISIS 539383 39 13 7 10 832 ISIS 539399 37 11 7 4 603 ISIS 539404 37 12 7 6 639 ISIS 539416 33 11 6 9 601 ISIS 539432 44 14 9 10 810 ISIS 539433 38 12 7 9 742

Organ Weights

Liver, heart, spleen and kidney weights were measured at the end of the study, and are presented in Table 228. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 228 Organ weights (g) Heart Liver Spleen Kidney PBS 0.35 3.6 0.2 0.8 ISIS 523723 0.31 4.9 0.7 0.8 ISIS 523789 0.34 4.8 0.6 0.8 ISIS 532254 0.32 5.0 0.6 1.0 ISIS 532401 0.32 3.8 0.4 0.8 ISIS 532420 0.29 4.6 0.7 1.0 ISIS 533178 0.34 5.2 0.7 0.9 ISIS 533234 0.30 4.4 0.6 1.0 ISIS 533932 0.31 3.9 0.5 0.9 ISIS 539376 0.29 4.4 0.4 0.8 ISIS 539380 0.31 6.3 1.6 1.2 ISIS 539383 0.31 4.5 0.6 1.0 ISIS 539399 0.31 4.5 0.8 1.0 ISIS 539404 0.34 4.9 0.6 1.0 ISIS 539416 0.32 4.7 0.7 0.9 ISIS 539432 0.30 3.8 0.4 0.8 ISIS 539433 0.28 4.1 0.7 1.0

Example 135 Tolerability of Deoxy, MOE, and (S)-cEt Gapmers Targeting Human GHR in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (50 mg/kg weekly dose). Two groups of rats were injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT and AST were measured and the results are presented in Table 229 expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in Table 229 expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 229 Liver function markers in Sprague-Dawley rats ALT AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 34 56 0.08 PBS 37 54 0.09 ISIS 541881 53 77 0.12 ISIS 542051 61 96 0.09 ISIS 542101 64 214 0.10 ISIS 542112 46 72 0.10 ISIS 542118 42 60 0.08 ISIS 542125 39 67 0.10 ISIS 542127 56 75 0.12 ISIS 542128 45 71 0.12 ISIS 542153 44 69 0.11 ISIS 542185 44 93 0.09 ISIS 542186 51 107 0.12 ISIS 545439 41 73 0.10 ISIS 545447 103 114 0.10 ISIS 541262 106 133 0.12 ISIS 541742 56 102 0.11 ISIS 541767 53 69 0.09 ISIS 541875 70 133 0.08

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 230, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 230 Kidney function markers (mg/dL) in Sprague-Dawley rats BUN Creatinine PBS 16 0.2 PBS 15 0.2 ISIS 541881 22 0.3 ISIS 542051 18 0.2 ISIS 542101 22 0.3 ISIS 542112 18 0.2 ISIS 542118 18 0.3 ISIS 542125 18 0.3 ISIS 542127 19 0.3 ISIS 542128 18 0.3 ISIS 542153 17 0.3 ISIS 542185 19 0.3 ISIS 542186 19 0.3 ISIS 545439 16 0.2 ISIS 545447 16 0.2 ISIS 541262 21 0.4 ISIS 541742 19 0.2 ISIS 541767 15 0.2 ISIS 541875 16 0.2

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 231. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 231 Hematology markers in Sprague-Dawley rats HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10⁶/μL) (10³/μL) (10³/μL) PBS 43 14 7 7 775 PBS 49 15 8 8 1065 ISIS 541881 41 13 8 6 553 ISIS 542051 39 13 7 9 564 ISIS 542101 37 12 7 15 603 ISIS 542112 45 14 8 10 587 ISIS 542118 47 15 8 7 817 ISIS 542125 41 13 7 7 909 ISIS 542127 44 14 8 10 872 ISIS 542128 44 14 8 7 679 ISIS 542153 48 15 8 7 519 ISIS 542185 44 14 8 9 453 ISIS 542186 44 14 8 12 433 ISIS 545439 40 12 7 11 733 ISIS 545447 43 13 8 9 843 ISIS 541262 46 14 8 17 881 ISIS 541742 47 15 8 10 813 ISIS 541767 53 16 9 9 860 ISIS 541875 42 13 7 9 840

Organ Weights

Liver, heart, spleen and kidney weights were measured at the end of the study, and are presented in Table 232. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 232 Organ weights (g) Heart Liver Spleen Kidney PBS 0.4 3.7 0.2 0.9 PBS 0.3 3.2 0.2 0.7 ISIS 541881 0.4 3.4 0.4 0.9 ISIS 542051 0.4 3.8 0.4 1.0 ISIS 542101 0.3 4.2 0.6 1.1 ISIS 542112 0.3 3.7 0.4 0.8 ISIS 542118 0.4 3.6 0.2 0.8 ISIS 542125 0.4 3.7 0.3 1.1 ISIS 542127 0.3 4.2 0.3 0.8 ISIS 542128 0.3 3.5 0.3 0.8 ISIS 542153 0.3 3.5 0.3 0.8 ISIS 542185 0.4 3.8 0.4 0.9 ISIS 542186 0.3 3.8 0.6 0.9 ISIS 545439 0.4 4.1 0.3 0.9 ISIS 545447 0.4 3.4 0.3 1.1 ISIS 541262 0.3 3.4 0.3 2.0 ISIS 541742 0.3 3.8 0.3 0.8 ISIS 541767 0.3 3.4 0.2 0.8 ISIS 541875 0.3 5.2 0.4 1.0

Example 136 Effect of ISIS Antisense Oligonucleotides Targeting Human GHR in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide efficacy and tolerability, as well as their pharmacokinetic profile in the liver and kidney, were evaluated.

At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (GENBANK Accession No. NW_001120958.1 truncated from nucleotides 4410000 to 4720000, designated herein as SEQ ID NO: 2332). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide to SEQ ID NO: 2332 is presented in Table 233. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence.

TABLE 233 Antisense oligonucleotides complementary to the rhesus GHR genomic sequence (SEQ ID NO: 2332) Target Target Start Stop SEQ ID ISIS No Site Site Chemistry NO 523723 149071 149090 5-10-5 MOE 918 532254 64701 64720 5-10-5 MOE 479 532401 147560 147579 5-10-5 MOE 703 541767 152700 152715 Deoxy, MOE 1800 and (S)-cEt 541875 210099 210114 Deoxy, MOE 1904 and (S)-cEt 542112 146650 146665 Deoxy, MOE 2122 and (S)-cEt 542118 149074 149089 Deoxy, MOE 2127 and (S)-cEt 542185 245782 245797 Deoxy, MOE 2194 and (S)-cEt

Treatment

Prior to the study, the monkeys were kept in quarantine during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 4 kg. Nine groups of 5 randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into the intracapsular region and outer thigh of the monkeys. The monkeys were dosed three times (days 1, 4, and 7) for the first week, and then subsequently once a week for 12 weeks with 40 mg/kg of ISIS oligonucleotide. A control group of 5 cynomolgus monkeys was injected with PBS in a similar manner and served as the control group.

During the study period, the monkeys were observed twice daily for signs of illness or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or illness was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. Scheduled euthanasia of the animals was conducted on day 86 by exsanguination after ketamine/xylazine-induced anesthesia and administration of sodium pentobarbital. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Hepatic Target Reduction RNA Analysis

On day 86, RNA was extracted from liver, white adipose tissue (WAT) and kidney for real-time PCR analysis of measurement of mRNA expression of GHR. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. ‘n.d.’ indicates that the data for that particular oligonucleotide was not measured. As shown in Table 234, treatment with ISIS antisense oligonucleotides resulted in significant reduction of GHR mRNA in comparison to the PBS control. Specifically, treatment with ISIS 532401 resulted in significant reduction of mRNA expression in all tissues.

TABLE 234 Percent inhibition of GHR mRNA in the cynomolgus monkey liver relative to the PBS control ISIS No Liver Kidney WAT 532401 60 47 59 532254 63 65 n.d. 523723 38 0 n.d. 542112 61 60 36 542118 0 22 27 542185 66 53 n.d. 541767 0 14 n.d. 541875 34 77 n.d.

Protein Analysis

Approximately 1 mL of blood was collected from all available animals at day 85 and placed in tubes containing the potassium salt of EDTA. The tubes were centrifuged (3000 rpm for 10 min at room temperature) to obtain plasma. Plasma levels of IGF-1 and GH were measured in the plasma. The results are presented in Table 235. The results indicate that treatment with ISIS oligonucleotides resulted in reduced IGF-1 protein levels.

TABLE 235 Plasma protein levels in the cynomolgus monkey IGF-1 (% GH baseline) (ng/mL) PBS 121 19 532401 57 39 532254 51 26 523723 77 16 542112 46 48 542118 97 6 542185 59 32 541767 101 22 541875 45 47

Tolerability Studies Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights were measured on day 84 and are presented in Table 236. Organ weights were measured on day 86 and the data is also presented in Table 236. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 532401 was well tolerated in terms of the body and organ weights of the monkeys.

TABLE 236 Final body and organ weights in cynomolgus monkey Spleen Kidney Liver Body Wt (kg) (g) (g) (g) PBS 2.7 2.8 12.3 56.7 532401 2.6 4.0 11.5 58.5 532254 2.6 4.8 15.4 69.5 523723 2.8 3.1 14.8 69.4 542112 2.6 3.5 13.6 60.0 542118 2.7 2.7 11.9 58.6 542185 2.6 5.5 17.2 68.5 541767 2.8 5.1 11.7 65.1 541875 2.8 5.5 13.2 55.0

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, blood samples were collected from all the study groups. The blood samples were collected via femoral venipuncture, 48 hrs post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes containing K2-EDTA anticoagulant, which were centrifuged to obtain plasma. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and AST and bilirubin were measured. The results indicate that antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 532401 was well tolerated in terms of the liver function in monkeys.

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, blood samples were collected from all the study groups. The blood samples were collected via femoral venipuncture, 48 hrs post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes containing K2-EDTA anticoagulant, which were centrifuged to obtain plasma. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).

The plasma chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 532401 was well tolerated in terms of the kidney function of the monkeys.

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 1.3 mL of blood was collected from each of the available study animals in tubes containing K2-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer (Bayer, USA).

The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose. Specifically, treatment with ISIS 532401 was well tolerated in terms of the hematologic parameters of the monkeys.

C-Reactive Protein Level Analysis

To evaluate any inflammatory effect of ISIS oligonucleotides in cynomolgus monkeys, blood samples were taken for analysis. The monkeys were fasted overnight prior to blood collection. Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C-reactive protein (CRP), which is synthesized in the liver and which serves as a marker of inflammation, was measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The results indicate that treatment with ISIS 532401 did not cause inflammation in monkeys.

Example 137 Measurement of Viscosity of ISIS Antisense Oligonucleotides Targeting Human GHR

The viscosity of select antisense oligonucleotides from the study described in the Examples above was measured with the aim of screening out antisense oligonucleotides which have a viscosity more than 40 cP. Oligonucleotides having a viscosity greater than 40 cP would be too viscous to be administered to any subject.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50° C. Part of (75 μL) the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in Table 237 and indicate that all the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above.

TABLE 237 Viscosity of ISIS antisense oligonucleotides targeting human GHR ISIS Viscosity No. Chemistry (cP) 523723 5-10-5 MOE 8 532254 5-10-5 MOE 22 532401 5-10-5 MOE 12 541767 Deoxy, MOE 13 and (S)-cEt 541875 Deoxy, MOE 33 and (S)-cEt 542112 Deoxy, MOE 10 and (S)-cEt 542118 Deoxy, MOE 14 and (S)-cEt 542185 Deoxy, MOE 17 and (S)-cEt

Example 138 Effect of ISIS Oligonucleotides Conjugated with GalNAc₃-7 vs. Unconjugated in a Mouse Model

ISIS oligonucleotides targeting murine GHR and that were either unconjugated or conjugated with GalNAc₃-7 were tested in BALB/c mice for efficacy and tolerability. BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing.

The oligonucleotides are all 5-10-5 MOE gapmers, which are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the murine gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to murine GHR mRNA, designated herein as SEQ ID NO: 2333 (GENBANK Accession No. NM_010284.2). The oligonucleotides are described in detail in the Table below.

TABLE 238 ISIS antisense oligonucleotides targeting murine GHR and conjugated with GalNAc3-7 or unconjugated Target SEQ ISIS Start ID No. Sequence Conjugated Site NO 563179 TGCCAACTCACTTGGATGTC No 772 2334 739949 TGCCAACTCACTTGGATGTC Yes 772 2334 563223 GAGACTTTTCCTTGTACACA No 3230 2335 706937 GAGACTTTTCCTTGTACACA Yes 3230 2335

Treatment

Two groups of seven-week old female BALB/c mice were injected subcutaneously for 4 weeks with 10 mg/kg/week, 25 mg/kg/week, or 50 mg/kg/week of ISIS 563223 or ISIS 563179. Two groups of seven-week old female BALB/c mice were injected subcutaneously for 4 weeks with 1 mg/kg/week, 5 mg/kg/week, or 10 mg/kg/week of ISIS 706937 or ISIS 739949. One group of female BALB/c mice was injected subcutaneously for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Target Reduction

To evaluate the efficacy of the ISIS oligonucleotides, plasma IGF-1 levels and mRNA expression levels of IGF-1 and GHR in liver, as well as mRNA expression levels of GHR in fat and kidney tissues, were measured. The results are presented in the Tables below.

The results indicate that the GalNAc3-7-conjugated oligonucleotides, ISIS 706937 and ISIS 739949, are 7-8 times more potent than the parent oligonucleotides with the same sequence, ISIS 563223 and ISIS 563179, in reducing GHR liver mRNA levels and were 6- to 8-fold more potent in reducing liver and plasma IGF-1 levels. Expression of GHR levels in the kidney and fat tissues were not decreased with GalNAc3-7-conjugated oligonucleotides, since the GalNAc3-7 conjugate group targeted the oligonucleotide specifically to the liver. This loss in fat and kidney reduction with GalNAc3-7-conjugated oligonucleotides did not affect reduction of IGF-1.

TABLE 239 Liver mRNA expression levels (% inhibition) at week 4 mg/kg/wk GHR ED₅₀ IGF-1 ED₅₀ ISIS 563223 10 62 4.2 15 19.4 25 97 69 50 99 77 ISIS 706937 1 59 0.6 24 3.4 5 97 63 10 98 69 ISIS 563179 10 50 9.6 22 49.4 25 67 31 50 93 50 ISIS 739949 1 39 1.2 18 6.4 5 89 57 10 94 45

TABLE 240 Plasma IGF-1 levels (% inhibition) at week 4 mg/kg/wk Week 2 Week 4 PBS — 0 0 ISIS 563223 10 13 22 25 40 60 50 43 71 ISIS 706937 1 20 31 5 46 64 10 61 67 ISIS 563179 10 19 25 25 10 24 50 25 46 ISIS 739949 1 11 24 5 29 41 10 37 31

TABLE 241 GHR mRNA expression levels (% inhibition) in fat and kidney at week 4 mg/kg/wk Fat Kidney ISIS 563223 10 21 45 25 30 66 50 62 65 ISIS 706937 1 0 5 5 0 0 10 0 14 ISIS 563179 10 4 38 25 14 40 50 20 41 ISIS 739949 1 4 11 5 0 1 10 0 8

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, glucose, cholesterol, and triglycerides were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, Calif.). The results are presented in the Table below. None of the ISIS oligonucleotides caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides. The GalNAc3-7-conjugated oligonucleotides had a slightly improved profile over the parent oligonucleotides.

TABLE 242 Plasma chemistry markers in BALB/c mice plasma at week 4 ALT AST Bilirubin Glucose Cholesterol Triglycerides mg/kg/wk (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) PBS — 26 58 0.2 165 70 123 ISIS 563223 10 23 69 0.3 157 74 186 25 39 91 0.3 165 62 160 50 49 118 0.3 159 56 115 ISIS 706937 1 25 62 0.2 152 64 167 5 28 64 0.2 180 53 140 10 27 65 0.2 165 56 133 ISIS 563179 10 28 78 0.4 156 65 131 25 28 95 0.2 152 59 118 50 63 108 0.3 157 80 143 ISIS 739949 1 24 66 0.2 156 66 114 5 29 80 0.2 153 76 161 10 31 59 0.3 174 78 155

The results taken together indicate that oligonucleotides targeting GHR mRNA expression when conjugated with GalNAc3-7 had tenfold greater potency and similar or improved tolerability profiles compared to the parent oligonucleotides.

Example 139 Tolerability Study of an ISIS Oligonucleotide Conjugated with GalNAc3-7 and Targeting Human GHR in Mice

ISIS 766720 was designed with the same sequence as ISIS 532401, a potent and tolerable oligonucleotide targeting human GHR and described in the studies above. ISIS 766720 is a 5-10-5 MOE gapmer with mixed backbone chemistry and conjugated with GalNAc3-7. The chemical structure of ISIS 766720 without the GalNAc3-7 conjugate group is denoted as mCes mCes Aeo mCeo mCes Tds Tds Tds Gds Gds Gds Tds Gds Ads Ads Teo Aeo Ges mCes Ae (SEQ ID NO: 703) and is fully denoted as:

Treatment

Groups of six-week old male CD-1 mice were injected subcutaneously for 6 weeks with 25 mg/kg/week, 50 mg/kg/week, or 100 mg/kg/week of ISIS 766720. One group of mice was injected subcutaneously for 6 weeks (days 1, 5, 15, 22, 29, 36, and 43) with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS 766720 on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. ISIS 766720 did not cause changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides and was deemed very tolerable.

TABLE 243 Plasma chemistry markers in CD-1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN mg/kg/wk (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS — 44 79 0.3 0.2 29 ISIS 25 29 47 0.2 0.2 34 766720 50 38 56 0.2 0.2 35 100 29 45 0.2 0.2 31

Body And Organ Weights

Body and organ weights were measured at the end of the study. The results are presented in the Table below. ISIS 766720 did not cause changes in weights outside the expected range for antisense oligonucleotides and was deemed very tolerable.

TABLE 244 Weights of CD-1 mice at week 6 Body Liver Kidney Spleen mg/kg/wk (g) (% body) (% body) (% body) PBS — 40 3.0 1.0 0.2 25 41 3.4 0.8 0.2 ISIS 766720 50 41 3.3 0.8 0.2 100 40 4.8 0.8 0.2 

1.-192. (canceled)
 193. A compound consisting of a conjugate group and a modified oligonucleotide according to the following formula:


194. A composition comprising the compound of claim 193 or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
 195. A prodrug comprising the compound of claim
 193. 196. (canceled)
 197. (canceled)
 198. A method comprising administering to an animal the compound claim
 193. 199. The method of claim 198, wherein the animal is a human.
 200. A method of treating a disease associated with excess growth hormone in a human comprising administering to the human a therapeutically effective amount of the compound of claim 193 thereby treating the disease associated with excess growth hormone.
 201. The method of claim 200, wherein the disease associated with excess growth hormone is acromegaly.
 202. The method of claim 200, wherein the treatment reduces IGF-1 levels.
 203. A method of preventing a disease associated with excess growth hormone in a human comprising administering to the human a therapeutically effective amount of a compound of claim 193 thereby preventing the disease associated with excess growth hormone.
 204. The method of claim 203, wherein the disease associated with excess growth hormone is acromegaly.
 205. The method of claim 200, comprising co-administering the compound and a second agent.
 206. The method of claim 205, wherein the compound or composition and the second agent are administered concomitantly.
 207. A method of reducing growth hormone receptor (GHR) levels in a human comprising administering to the human a therapeutically effective amount of the compound of claim 193, thereby reducing GHR levels in the human.
 208. The method of claim 207, wherein the human has a disease associated with excess growth hormone.
 209. The method of claim 207, wherein the disease associated with excess growth hormone is acromegaly.
 210. The method of claim 209, comprising co-administering the compound and a second agent.
 211. The method of claim 210, wherein the compound and the second agent are administered concomitantly.
 212. (canceled)
 213. (canceled) 